WO2016210223A1 - Immune modulation and treatment of solid tumors with antibodies that specifically bind cd38 - Google Patents

Immune modulation and treatment of solid tumors with antibodies that specifically bind cd38 Download PDF

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Publication number
WO2016210223A1
WO2016210223A1 PCT/US2016/039165 US2016039165W WO2016210223A1 WO 2016210223 A1 WO2016210223 A1 WO 2016210223A1 US 2016039165 W US2016039165 W US 2016039165W WO 2016210223 A1 WO2016210223 A1 WO 2016210223A1
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Prior art keywords
seq
antibody
specifically binds
cell
cells
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PCT/US2016/039165
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French (fr)
Inventor
Ahmadi TAHAMTAN
Tineke CADNEUF
Henk M. LOKHORST
Tina MUTIS
Amy SASSER
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Janssen Biotech, Inc.
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Publication date
Priority to JP2017566768A priority Critical patent/JP7041519B2/en
Priority to CA2990620A priority patent/CA2990620A1/en
Application filed by Janssen Biotech, Inc. filed Critical Janssen Biotech, Inc.
Priority to EP16815351.8A priority patent/EP3313441B1/en
Priority to MYPI2017704935A priority patent/MY193727A/en
Priority to CN201680048975.9A priority patent/CN107921120A/en
Priority to UAA201800631A priority patent/UA124799C2/en
Priority to AU2016281717A priority patent/AU2016281717B2/en
Priority to KR1020187001612A priority patent/KR20180012864A/en
Priority to BR112017027990-8A priority patent/BR112017027990A2/en
Priority to EA201890131A priority patent/EA037548B1/en
Priority to FIEP16815351.8T priority patent/FI3313441T3/en
Priority to MX2018000261A priority patent/MX2018000261A/en
Priority to TW105134914A priority patent/TWI742008B/en
Publication of WO2016210223A1 publication Critical patent/WO2016210223A1/en
Priority to IL256248A priority patent/IL256248A/en
Priority to PH12017502311A priority patent/PH12017502311A1/en
Priority to CONC2017/0013332A priority patent/CO2017013332A2/en
Priority to ZA2018/00476A priority patent/ZA201800476B/en
Priority to HK18114045.6A priority patent/HK1254942A1/en
Priority to JP2022015442A priority patent/JP2022070892A/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to methods of immune modulation and treatment of solid tumors with antibodies that specifically bind CD38.
  • the immune system is tightly controlled by a network of costimulatory and co- inhibitory ligands and receptors. These molecules provide secondaiy signals for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection and tumors, while limiting immunity to self (Wang et al, (Epub Mar. 7, 201 ⁇ ) JExp Med 208(3):577-92; Lepenies et al , (2008) Endocr Metah Immune Disord Drug Targets 8:279-288).
  • YERVOY J ipilimurnab
  • KEYTRUDA* pembrolizumab
  • OPDIVO* 1 nivolumab
  • NK natural killer cells
  • DC dendritic cells
  • effector T cells are capable of driving potent anti -tumor responses
  • tumor cells often induce an
  • immunosuppressive microenvironment favoring the development of immunosuppressive populations of immune cells, such as myeloid-derived suppressor cells (MDSC), regulatory T-cells (Treg) or regulatory B-ceils (Breg), which contribute to tumor immune tolerance and the failure of immunotherapy regimens in cancer patients and experimental tumor models.
  • immune cells such as myeloid-derived suppressor cells (MDSC), regulatory T-cells (Treg) or regulatory B-ceils (Breg), which contribute to tumor immune tolerance and the failure of immunotherapy regimens in cancer patients and experimental tumor models.
  • MDSC myeloid-derived suppressor cells
  • Treg regulatory T-cells
  • Breg regulatory B-ceils
  • the invention provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38.
  • the invention also provides a method for treating a patient having a regulator ' T- cell (Treg) mediated disease comprising administering to the patient in need thereof a therapeutically effecti ve amount of an antibody that specifically binds CDS 8.
  • Treg regulator ' T- cell
  • the invention also provides a method for treating a patient having a myeloid- derived suppressor cell (MDSC) mediated disease comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8.
  • MDSC myeloid- derived suppressor cell
  • the invention also provides a method for treating a patient having a regulatory B ⁇ cell (Breg) mediated disease comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8.
  • a regulatory B ⁇ cell (Breg) mediated disease comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8.
  • the invention also provides a method of suppressing activity of a regulatory T-ceil (Treg), comprising contacting the Treg with an antibody that specifically binds CDS 8.
  • Treg regulatory T-ceil
  • the invention also provides method of suppressing activity of a myeloid-derived suppressor cell (MDSC), comprising contacting the MDSC with an antibody that
  • CDS 8 specifically binds CDS 8.
  • the invention also provides a method of suppressing activity of a regulatory B- cell (Breg), comprising contacting the Breg with an antibody that specifically binds CD38.
  • a regulatory B- cell comprising contacting the Breg with an antibody that specifically binds CD38.
  • the invention also provides a method of enhancing an immune response in a patient, comprising administering to the patient an antibody that specifically binds CDS 8.
  • the invention also provides a method of treating a patient having a solid tumor comprising reducing the number of Tregs cells in the patient by administering to the patient an antibody that specifically binds CDS 8.
  • the invention also provides a method of treating a patient having a solid tumor, comprising reducing the number of myeloid-deri ved suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds CD38.
  • MDSC myeloid-deri ved suppressor cells
  • the invention also provides a method of of suppressing activity of an immune suppressor cell, comprising contacting the immune suppressing cell with an antibody that specifically binds CDS 8.
  • the invention also provides a method of treating a patient having a viral infection, comprising administering to the patient in need thereof an antibody that specifically binds CDS 8. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows that the median number of lymphocytes was increased in patients responding to DARZALEXTM (daratumumab) treatment at 8 mg kg (upper line) or 16 ltig/kg (Sower line) doses over time, and that the lymphocyte numbers returned to baseline after end of treatment.
  • X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (C lDl : cycle I, day 1: C1D4; cycle 1, day 4, etc).
  • SCR baseline; EOT: end of treatment; WK: week; POST-WK: post-treatment at indicated weeks; post-PD FU: follow-up after progression.
  • the highlighted areas in gray shades indicate the 25-27% Interquartile Range (IQR) for the data points for each visit for responders.
  • IQR Interquartile Range
  • Figure 2 shows the percent (%) change of absolute counts of CDS " T cells to baseline in peripheral blood in patients treated with DARZALEXTM (daratumumab) for each individual patient (light gray lines).
  • the X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (ClDl : cycle 1, day 1; C1D4; cycle 1, day 4, etc).
  • WK week; POST-WK: post-treatment at indicated weeks;
  • POST-PD FU follow-up after progression.
  • the black line shows the median % change for all patients.
  • Figure 3 shows the percent (%) change of absolute counts of CD4 + T cells to baseline in peripheral blood in patients treated with DARZALEX [M (daratumumab) for each individual patient (light gray lines).
  • the X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (Cl Dl : cycle 1, day 1 ; CID4; cycle 1 , day 4, etc).
  • the black line shows the median % change for ail patients.
  • Figure 4 shows the percent (%) change of absolute counts of CD8 + T cells to baseline in peripheral blood in patients treated with DARZALEXTM (daratumumab) for each individual patient (light gray lines).
  • the X-axis indicates time expressed as ireatmeni cycle and days of dosing within each treatment cycle (ClDl : cycle 1, day 1; C 1D4; cycle 1, day 4, etc).
  • WK week; Pre-PD FU: follow-up before progression; Post-PD FU: follow-up after progression.
  • the black line shows the median % change for all patients.
  • Figure 5 shows that the number of CD45XD3 + cells (measured as percentage of
  • lymphocytes in bone marrow aspirates was increased during DARZALEX lM (daratumumab) treatment ove time at doses 8 mg/kg or 16 mg/kg.
  • the graph includes both responders and non-responders as indicated.
  • the X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (C2D22: cycle 2, day 22; etc).
  • the highlighted areas in gray shade indicate the 25-27% Interquartile Range (IQR) for the data points for each visit for the non-responders dosed at 8 mg/kg, the responders dosed at 16 mg/kg, or the non -responders dosed at 16 mg/kg, respectively.
  • NR no-responder
  • R responder.
  • Figure 6 shows that the number of CD45 r CD3 + CD8 1 cells (measured as percentage of lymphocytes) in bone marrow aspirates was increased during DARZALEX lM (daratumumab) treatment over time at doses 8 mg/kg or 16 mg/kg.
  • the graph includes both responders and non-responders as indicated.
  • the X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (C2D22: cycle 2, day 22; etc).
  • SCR baseline; Post-PD FU 1 : follow-up after progression.
  • the highlighted areas in gray shade indicate the 25-27% Interquartile Range (IQR) for the data points for each visit for the non-responders dosed at 8 mg/kg, the responders dosed at 16 mg/kg, or the non-responders dosed at 16 mg/kg, respectively.
  • IQR Interquartile Range
  • Figure 7 A shows that the ratio of CD87 Treg and CD87CD4 + cells in peripheral blood expressed as median values of all treated patients increased over time during DARZALEX 1 * 1 (daratumumab) treatment.
  • SRC baseline.
  • Figure 7B shows that the ratio of CD87Treg cells in bone marrow aspirates expressed as median values of all treated patients increased over time during DARZALEX lM
  • Figure 8A shows that responders had increased CD8 + T-cell cionality when compared to non-responders, as measured using % change in abundance (CIA) of particular clonal cells.
  • Figure 8B shows the fold change in CD8 + T-cell cionality in individual patients pre- vs. post DARZALEX lM (daratumumab) treatment. Responders are indicated with the star. Cionality was measured as fold change in abundance (CIA) of particular clonal cells. Study: GEN501 17 patient subset.
  • Figure 8C shows that responders (Group A) had greater total expansion in the TCR repertoire, measured using CIA (change in abundance) when compared to non-responders (Group B). P 0.037. Study: GEN501 17 patient subset.
  • Figure 8E shows the maximum CIA of a single T-cell clone in responders (Group A) and non-responders (Group B). Study : GEN501 17 patient subset.
  • Figure 8F shows that responders (Group A) had greater maximum expansion of a single clone, measured using maximum % CIA, when compared to non -responders (Group B). P 0.0477. Study: GEN501 17 patient subset.
  • Figure 9A shows the percentage (%) of CD8 + naive ceils in peripheral blood in non- responders (NR, black squares) and in patients having at least minimal response (MR, white squares) to DARZALEX IM (daratumumab) at baseline, or at 2 weeks, 4 weeks or 8 weeks of treatment, or after relapse.
  • Study: GEN501 17 patient subset. **p 1.82 x 10 "4 .
  • Figure 9B shows the percentage of CD8 + central memory cells (Tern) in peripheral blood in non-responders (NR, black squares) and in patients having at least minimal response (MR, white squares) to DARZALEX lM (daratumumab) at baseline, or at 2 weeks, 4 weeks or 8 weeks of treatment, or after relapse.
  • Study: GENS 01 17 patient subset. *p .88xl0 ⁇ .
  • Figure 9C shows the percentage increase of HLA Class I restricted CD3 ⁇ 4"r T cells in peripheral blood at baseline, or at week 1 , 4 or 8 of treatment, or after relapse.
  • Figure 9D shows that CDS 8 is expressed at low levels in CD8 + naive T cells and in CD8 " central memory cells (Tern) in peripheral, blood at bassline or on treatment.
  • Figure 10A shows a histogram of FACS analyses showing frequency of Tregs (CD3 T CD3 + CD4 + CD25 + CD127 d,tn (top histogram, P4 cell population) and the frequency of CD38 Tregs within the ' Treg population (bottom histogram, P5 cell population) in multiple myeloma patients at baseline.
  • Study: GEN501 17 patient subset- Figure 10B shows a histogram of FACS analyses showing frequency of Tregs (CD3 " CD3 " CD4 4" CD25 "t CD127 dim (top histogram, P4 cell population) and the frequency of CD38 " Tregs within the Treg population (bottom histogram, P5 cell population) in multiple myeloma patients after DARZALEX lM (daratumumab) treatment. DARZALE lM (daratumumab) treatment depleted CD38 " Tregs. Study: GEN501 17 patient subset.
  • Figure IOC shows that frequency of the CD38 M h CD3 ⁇ CD4 i CD25 + CD127 dils Tregs in patients treated with DARZALEXTM (daratumumab) at baseline, or at 1 week, 4 week, 8weeks, after relapse or at end of treatment at 6 months (EOT), CD38 tasa Treg frequency was reduced with DARZALEX lM (daratumumab) treatment and returned to baseline at EOT.
  • Figure 10D shows the CD87Treg cell ratio in responders and non-responders at baseline, at 1 week, 4 weeks and 8 weeks of treatment.
  • Figure 10E shows that effector cell proliferation is inhibited more efficiently in the presence of CD38 r Tregs when compared to the CD38 ⁇ Tregs or negative controls. Error bars represent standard error. Asterisks denote significant changes. Samples were obtained from multiple healthy donors. Cell proliferation was assessed through the dilution of carboxyfluorescein succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein succinimidyl ester
  • FIG 11 shows that Myeloid-derived suppressor cells (MDSC) are present in multiple myeloma patients (top graph, boxed cells) and that about half of the cells expressed CD38 (middle graph, boxed cells).
  • the CD38high MDSC population was depleted in patients treated with one infusion of DARZALEX iM (daratumumab) (bottom graph, boxed cells).
  • Figure 12 shows that the number of CD38high MDSCs (CD1 1 b + HLADR " CD 14 "
  • CD33 + CD15 + was reduced in patients after 1 week, 4 week or 8 week treatment with DARZALEX J M (daratumumab) when compared to the baseline, and returned to close to baseline after end of treatment (EOT). Relapsed patients still demonstrated reduced CD38high MDSCs.
  • the vertical lines indicate the median values in each group.
  • Patients 2, 4, 15, 16 and 17 demonstrated high initial CD38high MDSCs population. Study: GEN501 17 patient subset.
  • Figure 13 shows that the patients with highest CD38high MDSCs (patients 2, 4, 15, 16 and 17) had the highest Progression-Free Survival (PFS). These patients had either partial Response (PR) or Minimal Response (MR) to DARZALEX [M (daratumumab) treatment. SD : Stable Disease; PD : Progressive Disease. X-axis shows the PFS for each individual numbered patient.
  • PR partial Response
  • MR Minimal Response
  • SD Stable Disease
  • PD Progressive Disease
  • X-axis shows the PFS for each individual numbered patient.
  • Figure 14 shows that MDSC are sensitive to DARZALEX lM (daratumumab)-induced ADCC. Daudi ceils were used as positive control for target ceils in the assays. % cell lysis was measured.
  • Figure ISA shows that CD38 + Bregs were depleted in patients treated with DARZALEX 1M (daratumumab) at Week 1, Week 4 and Week 8 of treatment.
  • Figure 15B shows that CD38 + Bregs secrete lL-10 upon stimulation.
  • Figure 16A shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN-y production in PBMCs from DARZALEX lM (daratumumab) treated patient with VGPR at baseline and at indicated times during treatment.
  • OD optical density.
  • White bar negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only.
  • Asterix indicates a statistically significant change.
  • Pre 4, 8, 10 Week 4, 8 or 10 of treatment.
  • Figure 16B shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN- ⁇ production in PBMCs from DA ZALEX lM (daratumumab) treated patient with CR at baseline and at indicated times during treatment.
  • OD optical density.
  • White bar negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only, Asterix indicates a statistically significant change.
  • Pre 4, 8, 10 Week 4, 8 or 10 of treatment.
  • Figure 16C shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN- ⁇ production in PBMCs from DA ZALEX lM (daratumumab) treated patient with PD at baseline and at indicated times during treatment.
  • OD optical density.
  • White bar negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only, Ns: not significant.
  • Figure 16D shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN- ⁇ production in PBMCs from DA ZALEX lM (daratumumab) treated patient with MR at baseline and at indicated times during treatment.
  • OD optical density.
  • White bar negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only. Ns: not significant.
  • Figure 16E shows the percentage (%) of proliferating virus-reactive T ceils in PBMCs from
  • DARZALEXTM (daratumumab) treated patient with VGPR at baseline and at indicated times during treatment.
  • White bar negative control; black bar: CEF added.
  • Asterix indicates a statistically significant change.
  • Pre 4, 8, 10 Week 4, 8 or 10 of treatment.
  • Figure 16F shows the percentage (%) of proliferating virus-reactive T cells in PBMCs from
  • DARZALEXTM (daratumumab) treated patient with CR at baseline and at indicated times during treatment.
  • White bar negative control; black bar: CEF added.
  • Asterix indicates a statistically significant change.
  • Pre 4, 8, 10 : : Week 4, 8 or 10 of treatment.
  • Figure 17A shows a histogram of FACS analyses showing CD38 expression levels on natural killer cells (NK), monocytes, B cells and T cells from a healthy donor.
  • NK natural killer cells
  • Figure 17B shows a histogram of FACS analyses showing CD38 expression levels on plasma cells, natural killer ceils (NK), monocytes, B cells and T cells from a multiple myeloma patient.
  • NK natural killer ceils
  • Figure 17C shows a comparison of the mean fluorescent intensity (MFI) of CD38 in CD38+ Tregs, Bregs, NK, B cells and T cells from relapsed and refractory multiple myeloma patients, CD38 was expressed at lower level in B cells and T cells when compared to the CD38+Tregs, Bregs and NK cells.
  • MFI mean fluorescent intensity
  • Figure 18 shows that PD-L1 protein is downregulated in PBMC samples from responders (R) and upregulated in non-responders (NR.) over time.
  • SD stable disease.
  • C1D1 cycle 1 day 1 ; C3D1, cycle 3, day 1.
  • Y axis shows the log2 protein concentration values.
  • timing for example, reference to “a cell” includes a combination of two or more cells, and the like .
  • CD38 refers to the human CD38 protein (synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1 , cyclic ADP-ribose hydrolase 1).
  • Human CD38 has an amino acid sequence shown in GenBank accession number NP 001766 and in SEQ ID NO: 1. It is well known that CD38 is a single pass type II membrane protein with amino acid residues 1-21 representing the cytosolic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain of CD38.
  • Antibodies as used herein is meant in a broad sense and includes
  • immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, hispecific or rnultispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the
  • immunoglobulin molecule that comprises an antigen binding site of the required
  • Immunoglobulins may be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes TgA l, IgA2, TgGl, IgG2, IgG3 and IgG4.
  • Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • Antibody fragments refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL).
  • HCDR heavy chain complementarity determining regions
  • LCDR light chain complementarity determining regions
  • VH heavy chain variable region
  • VL light chain variable region
  • Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single ann of an antibody; a domain antibody (dAb) fragment (Ward et a!., Nature 341:544- 6, 1989), which consists of a VH domain.
  • dAb domain antibody
  • VH and VL domains may be engineered and linked together via a synthetic linker to form various types of single chain antibody designs in which the VH/VL domains pair intramolecularly, or intennolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Intl. Pat. Publ. Nos.
  • scFv single chain Fv
  • diabody described for example in Intl. Pat. Publ. Nos.
  • Isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding CD38 is substantially free of antibodies that specifically bind antigens other than human CD38).
  • An isolated antibody that specifically binds CD38 may have cross-reactivity to other antigens, such as orthologues of human CD38, such as Macaco, fascicidaris (cynomolgus) CD38.
  • the bispecific antibody specifically binds two antigens of interest, and is substantially free of antibodies that specifically bind antigens other that the two antigens of interest.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • Isolated antibody encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.
  • Specific binding or “specifically binds” or “binds” refers to an antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens.
  • the antibody binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K D ) of about IxlO "8 M or less, for example about IxlO "9 M or less, about 1x10 "10 M or less, about lxl 0 " S 1 M or less, or about IxlO "12 M or less, typically with the K D that is at least one hundred fold less than its K D for binding to a nonspecific antigen (e.g., BSA, casein).
  • K D equilibrium dissociation constant
  • the dissociation constant may be measured using standard procedures.
  • Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Calhthrix jacchus (common marmoset, marmoset). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.
  • homologs such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Calhthrix jacchus (common marmoset, marmoset). While a monospecific antibody specifically binds one antigen or one epitope
  • An antibody variable region consists of a "framework" region interrupted by three "antigen binding sites".
  • the antigen binding sites are defined using various terms:
  • Complementarity Determining Regions three in the VH (HCDR1 , HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat (1970) J Exp Med 132:211-50: Kabat et a! Sequences of Proteins of Immunological Interest, 5th Ed.
  • Hypervanable regions three in the VH (HI , H2, H3) and three in the VL (L I, L2, L3) refer to the regions of antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk (1987) A:io/ /3 ⁇ 4o/ 196:901-17).
  • Other terms include “IMGT-CDRs” (Lefranc et al, (2003) Dev Comparat Immunol 27:55-77) and “Specificity Determining Residue Usage” (SDRU) (Almagro (2004) Mo I Recognit 17: 132-43).
  • IMGT International ImMunoGeneTics
  • Chothia residues as used herein are the antibody VL and VH residues numbered according to Al-Lazikani (AI-Lazikani et al, ( 1997) JMol Biol 273:927-48).
  • Framework or “framework sequences” are the remaining sequences of a variable region other than those defined to be antigen binding sites. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined.
  • Humanized antibody refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.
  • ''Human antibody refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin.
  • a human antibody comprises heavy or light chain variable regions that are "derived from” sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes.
  • Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci.
  • a "human antibody” may contain amino acid differences when compared to the human gennline immunoglobulin or rearranged immunoglobulin genes due to for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding site, or both.
  • human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in ammo acid sequence to an amino acid sequence encoded by human gennline immunoglobulin or rearranged immunoglobulin genes.
  • human antibody may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et at, (2000) JMol Biol 296:57-86, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et ai, (2010) JMol Biol 397:385-96, and Intl. Pat. Publ. No.
  • Human antibodies derived from, human immunoglobulin sequences may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody gennline repertoire in vivo.
  • Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of human antibody.
  • “Recombinant antibody” includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal, for example a mouse or a rat that is transgenic or transchromosomai for human
  • immunoglobulin genes or a hybridoma prepared therefrom (described further below), antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.
  • “Monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.
  • “Monoclonal antibody” therefore refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of Oterminai lysine from the antibody heavy chain.
  • Monoclonal antibodies may have heterogeneous glycosylation within the antibody population.
  • Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.
  • Epitope means a portion of an antigen to which an antibody specifically binds.
  • Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may- have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be composed of contiguous and/or noncontiguous amino acids that form a conformational spatial unit. For a noncontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3- dimensional space through the folding of the protein molecule.
  • V ariant refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.
  • “In combination with” means that two or more therapeutics are administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • “Treat” or “treatment” refers to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological change or disease, such as the development or spread of tumor or tumor ceils, or to provide a beneficial or desired clinical outcome during treatment.
  • Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, lack of metastasis, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • 'Treatment may also mean prolonging survival as compared to expected survival if a subject was not receiving treatment.
  • Those in need of treatment include those subjects already with the undesired physiological change or disease as well as those subjects prone to have the physiological change or disease.
  • “Therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual.
  • Exemplar ⁇ ' indicators of an effective therapeutic or combination of therapeutics include, for example, improved well-being of the patient, reduction in a tumor burden, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer ceils to other locations in the body.
  • “Inhibits growth” refers to a measurable decrease or delay in the tumor cell growth or tumor tissue in. vitro or in vivo when contacted with a therapeutic or a combination of therapeutics or drugs, when compared to the decrease or delay in the growth of the same tumor ceils or tumor tissue in the absence of the therapeutic or the combination of therapeutic drags. Inhibition of growth of a tumor cell or tumor tissue in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • Tregs refers to T lymphocytes that regulates the activity of other T cell(s) and/or other immune cells, usually by suppressing their activity.
  • the Tregs may be CD3 + CD4 + CD25 + CD127 dim T cells, it is appreciated that the Tregs may not be fully restricted to this phenotype, and may express Foxp3.
  • Effective T cells or "Teffs” or “Teff ' refers to T lymphocytes that cany out a function of an immune response, such as killing tumor cells and/or activating an antitumor immune-response which can result in clearance of the tumor cells from the body.
  • the Teffs may be CD3 + with CD4 T or CD8 + .
  • the Teffs may secrete, contain or express markers such as IFN-y, granzyme B and ICOS. It is appreciated that the Teffs may not he fully restricted to these phenotypes.
  • Treg function refers to a suppressive function of the Tregs that relates to regulation of host immune responses and/or prevention of autoimmunity.
  • Function of Tregs may be suppression of an anti-tumor response elicted by CD8 + T cells, natural killer (NK) cells, M0 cells, B cells, or dendritic cells (DCs), or suppression of proliferation of effector T cells.
  • 'Inhibit function of Tregs or “inhibit Treg function” refers to decreasing the level of function of Tregs in vitro or in vivo in an animal or human subject, which may be determined by conventional techniques known in the art.
  • the level of the function of Tregs may be decreased by, for example, at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.
  • “Inhibit function of Tregs” include reducing the number of Tregs, for example by killing the Tregs via antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Myeloid-derived suppressor cells or “MDSCs” or “MDSC” refers to a specialized population of cells that are of the hematopoietic lineage and express the macrophage/monocyte marker CD1 lb and the granulocyte marker Gr ⁇ l/Ly ⁇ 6G.
  • Phenotype of the MDSCs may be for example CD1 lb + HLA-DR " CD14 " CD33 + CD 15 + .
  • the MDSCs express low or undetectable expression of the mature antigen presenting cell markers MHC Class II and F480.
  • the MDSCs are immature cells of the myeloid lineage and may further differentiate into several cell types, including macrophages, neutrophils, dendritic cells, monocytes or granulocytes.
  • the MDSCs may be found naturally in normal adult bone marrow of human and animals or in sites of normal hematopoiesis, such as the spleen.
  • Inhibit function of MDSCs or “inhibit MDSC function” refers to decreasing the level of function of MDSCs in vitro or in vivo in an animal or human subject, which may be determined by conventional techniques known in the art.
  • the level of the function of MDSC may be decreased by, for example, at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.
  • “Inhibit function of MDSC” include reducing the number of MDSC, for example by killing the MDSC via antibody effector functions, such as ADCC.
  • the MDSCs may suppress T cell responses such as proliferation, clonal expansion or cytokine production by various mechanisms such as production of reactive oxygen species, peroxynitrites, increased arginase metabolism due to high levels of arginase, and increased nitrous oxide synthase.
  • the MDSCs may response to IFN- ⁇ and several cytokines such as IL-4 and IL-13.
  • IFN- ⁇ may activate MDSCs which induces the activity of nitric-oxide synthase 2 (NOS2).
  • NOS2 cytokines such as interleukin-4 (IL-4) and IL-13 may activate MDSCs which may lead to the induction of arginase-1 (ARG1) activity.
  • the metabolism of L-arginine by either NOS2 or ARG1 may lead to the inhibition of T-cell proliferation, and the activity of both enzymes together can result in T-cell apoptosis through the production of reactive nitrogen-oxide species.
  • Treg related disease refers to a disease or disorder linked to T regulatory cells (Tregs). Treg related disease may be caused by Treg function, for example, suppression of an anti-tumor response or suppression of effector T cell proliferation.
  • the Treg mediated disease may be cancer.
  • Treg related disease and “Treg mediated disease” are used exchangeable herein.
  • Enhance respon se of effector T cells or “enhance T cell responses” refers to enhancement or stimulation of effector T cells in vitro or in vivo in an animal or human subject to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T-celis.
  • Exemplary T-cell responses are proliferation, secretion of ⁇ - interferon from CD 8 + T-cells, antigen responsiveness, or clonal expansion. The manner of measuring this enhancement is known to one of ordinary skill in the art.
  • MDSC related disease refers to a disease or disorder linked to myeloid-derived suppressor cells (MDSCs). MDSC related disease may be caused by a MDSC function, for example, suppression of an anti -tumor response or effector T cell proliferation .
  • the MDSC mediated disease may be cancer.
  • MDSC related disease and MDSC mediated disease are used exchangeably herein.
  • Regular - B cell or “Breg” or “Bregs” refers to B lymphocytes that suppress immune responses.
  • the Bregs may be CD 19 D24 D38 + cells, and may suppress immune responses by inhibiting T cell proliferation mediated by IL-10 secreted by the Bregs. It is appreciated that other Breg subsets exists, and are described in for example Ding et a!., (2015) Human Immunology 76: 615-621.
  • Breg related disease refers to a disease or disorder linked to regulator ⁇ ' B cells. Breg related disease may be caused by for example Breg mediated suppression of an antitumor response or effector T cell proliferation.
  • the Breg mediated disease may be cancer.
  • “Breg related disease” and “Breg mediated disease” are used exchangeably herein.
  • the invention provides a method for treating a patient having a solid tumor with an antibody that specifically binds CDS 8 regardless whether the tumor cells express CDS 8 or not.
  • the invention further provides methods for treating a patient having regulatory T cell (Treg), myeloid-derived suppressor cell (MDSC) or regulatory B cell (Breg) mediated disease.
  • the invention further provides methods for modulating Treg, MDSC or Breg activity to treat solid tumors that are CDS 8 positive and/or associated with high levels of these immune suppressive cells.
  • the invention is based, at least in part, on the discover ⁇ - that the anti-CD38 antibody DARZALEX 1M (daratumumab) has an immunomodulatory activity in patients, reducing the number of immune suppressive Tregs, MDSCs and Bregs, increasing the number of CDS ' ' ' T cells and the ratio of CD8 " to Tregs, promoting CD8 + central memory cell formation and increasing T cell cionality.
  • DARZALEX 1M daratumumab
  • DARZALEX 1M (daratumumab) and other anti-CD38 antibodies are being evaluated in the clinic for their efficacy to treat heme malignancies and plasma cell disorders, including multiple myeloma, by the a bility of the antibody to eliminate CDS 8- positive cells by antibody effector functions, such as ADCC, CDC, ACDP and apoptosis, but their immunomodulatory activity in promoting adaptive immune responses has not been recognized.
  • Other immune modulator ⁇ ' antibodies (anti-PDl, anti-CTLA4) function through targeting components of the immune system that suppress anti-tumor responses.
  • anti-PDl antibodies have been demonstrated to increase T-cell proliferation, stimulate antigen-specific memory responses, and partially relieve Treg-mediated suppression of effector T cells in vitro (for example, see U.S. Pat. No. 8,779,105).
  • Two anti-PD-1 antibodies are currently approved for treatment of melanoma, OPDIVO® (nivolumab) and KEYTRUDA ⁇ (pembrolizumab) and these antibodies are in clinical development for various solid tumors, such as lung non-small cell carcinoma, prostate, head and neck, gastrointestinal, stomach, prostate, fallopian tube, ovarian, pancreatic, breast and brain cancer, renal, bladder, urethral, oesophageal and colorectal cancer.
  • Anti- CTLA-4 antibody YERVOY ⁇ (ipilimumab) has been approved for treatment of melanoma.
  • YERVOY® (ipilimumab) and another anti-CTLA-4 antibody, tremelimumab are also being developed for prostate, non-small cell lung cancer, ovarian, gastrointestinal, stomach, colorectal, renal, oesophageal, and genitourinary cancer.
  • DARZALEX 1M (daratumumab) described herein
  • the invention provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a regulatory T cell (Treg) mediated disease, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 for a time sufficient to treat the Treg mediated disease.
  • Treg regulatory T cell
  • the invention also provides for a method of treating a patient having a myeloid- derived suppressor cell (MDSC) mediated disease, comprising administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CDS 8 for a time sufficient to treat the MDSC mediated disease.
  • MDSC myeloid- derived suppressor cell
  • the invention also provides for a method of treating a patient having a regulatory B cell (Breg) mediated disease, comprising administering to the patient in need tliereof a therapeutically effective amount of an antibody that specifi cally binds CD38 for a time sufficient to treat the Breg mediated disease.
  • Breg regulatory B cell
  • the invention also provides for a method of suppressing activity of a regulatory T cell (Treg), comprising contacting the regulatory T cell with an antibody that specifically binds CD38,
  • the invention also provides for a method of suppressing activity of a myeloid- derived suppressor cell (MDSC), comprising contacting the MDSC with an antibody that specifically binds CD38.
  • MDSC myeloid- derived suppressor cell
  • the invention also provides for a method of suppressing activity of a regulator ⁇ ' B cell (Breg), comprising contacting the Breg with an antibody that specifically binds CDS 8.
  • a regulator ⁇ ' B cell comprising contacting the Breg with an antibody that specifically binds CDS 8.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising reducing the number of regulatory T ceils (Treg) in the patient by administering to the patient an antibody that specifically binds CD38.
  • Treg regulatory T ceils
  • the invention also provides for a method of treating a patient having a solid tumor, comprising reducing the number of myeloid -derived suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds CDS 8.
  • MDSC myeloid -derived suppressor cells
  • the invention also provides for a method of treating a patient having a solid tumor, comprising reducing the number of regulatory B cells (Breg) in the patient by- administering to the patient an antibody that specifically binds CDS 8.
  • the invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof an antibody that specifically binds CD38 for a time sufficient to enhance the immune response.
  • the patient has a viral infection.
  • the invention also provides for a method of treating a viral infection in a patient, comprising administering to the patient in need therefor an antibody that specifically binds CD38 for a time sufficient to treat the viral infection.
  • the immune response is an effector T cell (Teff) response.
  • the Teff response is mediated by CD4 + T cells or CD8 " T cells.
  • the Teff response is mediated by CD4 f T cells.
  • the Teff response is mediated by CD8 r T cells.
  • the Teff response is an increase in the number of CDS " T cells, increased CD8 + T cell proliferation, increased T cell clonal expansion, increased CD8 " memory cell formation, increased antigen -dependent antibody production, or increased cytokine, chemokine or interleukin production.
  • Proliferation of T cells may be assessed for example by measuring the rate of DNA synthesis using tritiated thymidine or measuring production of interferon -y (IFN- ⁇ ) in vitro, or measuring absolute number or percentage of T cells in a population of cells from patient samples using known methods.
  • IFN- ⁇ interferon -y
  • Clonal expansion may be assessed by for example sequencing TCR from a pool of T cells using know methods.
  • Memory cell formation may be assessed by measuring the ratio of naive T cells (CD45R07CD62L ) to memory T ceils (CD45RO + /CD62L gh ) using for example FACS.
  • Cytokine, chemokine or interleukin production such as production of interferon - ⁇ (IFN- ⁇ ), tumor necrosis factor-alpha (TNF-a), IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL- 12, IL 3, IL-10, IL-18 and IL-23, ⁇ - ⁇ , ⁇ - ⁇ , RANTES, CCL4 may be assessed using standard methods such as EL1SA or ELLISPOT assay.
  • Antigen-specific antibody production may be assessed from samples derived from patient using standard methods, such as ELISA or radioimmunoassay (RTA).
  • standard methods such as ELISA or radioimmunoassay (RTA).
  • the meaning of "increase” or “increasing” various Teff responses is readily understood.
  • the increase may be increase of at least about 5%, at least about 10%, 25%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400% or more in a test sample or in a subject when compared to control, e.g., for example in a patient treated with
  • the meaning of “reduce “ ' or “reducing” or “decreasing” or “decrease” the number of Tregs, MDSCs and/or Bregs is readily understood.
  • the decrease may be at least about 10%, 25%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 1 10%, 120%, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400% or more in a test sample or in a subject when compared to control,
  • the antibody that specifically binds CD 38 inhibits function of immune suppressor cells.
  • the immune suppressor cells are regulatory- T cells (Tregs), myeloid-derived suppressor cells (MDSC) or regulator ⁇ ' B cells (Bregs).
  • the Tregs are CD3 + CD4 + CD25 + CD127 d,tn T cells.
  • the CD3 r CD4 + CD25 + CD127 aiBI cells express Foxp3.
  • the CD3 + CD4 + CD25 ⁇ CD127 diin T cells express CD38.
  • Treg function such as their ability to suppress Teff cells, may be assessed using known methods, such as assessing the ability of Tregs to suppress Teff proliferation in mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • Treg function may be inhibited by for example reducing the relative number of Tregs when compared to Teffs (e.g. increasing the ratio of CDSVTreg cells) by direct killing of Tregs or a sub-population of Tregs, such as CD38 " Tregs.
  • the Treg function is inhibited by killing the Treg cells.
  • the Treg killing is mediated by antibody-induced antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or apoptosis mduced by an antibody specifically binding CD38.
  • ADCC antibody-induced antibody- dependent cell cytotoxicity
  • ADCP antibody-dependent cell phagocytosis
  • CDC complement-dependent cytotoxicity
  • the Treg killing is mediated by ADCC.
  • the CD38 + Tregs are killed.
  • Tn some embodiments, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of Tregs are killed.
  • CD38 is expressed only in a portion of Tregs and MDSCs, it is expected that treatment of patients with solid tumors will not result in systemic depletion of Tregs and MDSCs, therefore likely providing an improved safety profile.
  • the MDSCs are CD1 lbHLA-DR " CD14 " CD33 + CD15 + cells.
  • the CDl lb + HLA-DR-CD14 _ CD33 4' CD15 MDSCs express
  • MDSC function may be inhibited for example by reducing the number of MDSCs by direct killing of the cells.
  • the MDSC function is inhibited by killing the CD38
  • the MDSC killing is mediated by antibody-induced antibody-dependent cell cytotoxicity (ADCC), antibody -dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or apoptosis induced by the antibody that specifically binds CD38.
  • ADCC antibody-induced antibody-dependent cell cytotoxicity
  • ADCP antibody -dependent cell phagocytosis
  • CDC complement-dependent cytotoxicity
  • the MDSC killing is mediated by ADCC.
  • the Bregs are CD19 ⁇ CD24 + CD38 + cells.
  • the Breg function may be inhibited for example by reducing the number of Bregs by direct killing of the Bregs.
  • the Breg function is inhibited by killing the CD38 " Bregs.
  • the Breg killing is mediated by antibody-induced antibody- dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement -dependent cytotoxicity (CDC) or apoptosis induced by the antibody that specifically binds CD38,
  • the Breg killing is mediated by ADCC.
  • Tregs play a critical role in the maintenance of peripheral self-tolerance.
  • Naturally occurring CD4 T CD25 i " Tregs are produced in the thymus and express Foxp3, a transcriptional factor required for establishment and maintenance of Treg lineage identity and suppressor function.
  • Tregs can accumulate at a disease site (e.g. within tumor), where they suppress the effector function of tumor antigen specific T cells, resulting in insufficient anti-tumor responses.
  • Increased densities of tumor-infiltrating Foxp3 + Tregs have been associated with poor prognosis in various solid tumors, including pancreatic, ovarian, and hepatocellular carcinoma. Depletion of Tregs results in enhanced antitumor immunity and tumor rejection in murine models but may also result in the development of autoimmune diseases.
  • MDSC Myeloid-derived suppressor cells
  • NK natural killer cells
  • NKT natural killer T cells
  • CD8 + T cells CD8 + T cells. While the mechanism of NK cell inhibition is currently not well-understood, multiple pathways are responsible for MDSC-mediated T cell suppression including production of arginase 1/ARGl and upregulation of nitri c oxide synthase 2 (NOS2).
  • ARGI and NOS2 metabolize L-arginine and either together or separately blocks the translation of the T cell CD3zeta chain, inhibits T cell proliferation, and promotes T cell apoptosis. Additionally, MDSCs secrete immunosuppressive cytokines and induce regulatory T cell development.
  • MDSC are induced by pro-inflammatory cytokines and are found in increased numbers in infectious and inflammatory pathological conditions. They accumulate in the blood, bone marrow, and secondary lymphoid organs of tumor-bearing mice and their presence in the tumor microenvironment as been suggested to have a causative role in promoting tumor-associated immune suppression.
  • MDSC have been described in patients with colon carcinoma, melanoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, non-small cell lung carcinoma, renal cell carcmoma, pancreatic adenocarcinoma and breast carcinoma (Mandruzzato ei al, (2009) J Immunol 182: 6562-6568; Lm et al, (2009) J Cancer Res dm Oncol 136: 35-45; Ko ei al .
  • Diaz et al (Diaz-Montero ei at, (2009) Cancer Immunol Immunother 58: 49-59) propose that accumulation of MDSC correlates with more advanced disease and poor prognosis.
  • Tumor-infiltrating Bregs have been identified in solid tumors, and the Bregs may promote tumor growth and metastasis by various mechanisms such as suppressing the antitumor activity of CD8 " T ceils and NK cells, as described in for example Ding et al, (2015) Human Immunology 76:615-62.
  • Antibody-dependent cellular cytotoxicity is a mechanism for inducing cell death that depends upon the interaction of antibody -coated target cells with effector ceils possessing lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.
  • effector ceils possessing lytic activity such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.
  • FcyR Fc gamma receptors
  • NK cells express FcyRTIIa
  • monocytes express FcyRI, FcyRTI and FcvRITIa.
  • Death of the antibody-coated target cell such as CD38-expressing cells, occurs as a result of effector cell activity through the secretion of membrane pore-forming proteins and proteases.
  • the antibody may be added to CD38 -expressing cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell . Cytolysis is generally detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed ceils.
  • label e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins
  • exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.
  • PBMC peripheral blood mononuclear cells
  • target cells include Tregs or MDSCs expressing CD38.
  • target cells are labeled with 20 ⁇ of 3l Cr for 2 hours and washed extensively.
  • Cell concentration of the target cells may be adjusted to 1 ⁇ 10 6 cells/ml, and anti-CD38 antibodies at various concentrations are added.
  • Assays are started by adding target cells at an effectontarget cell ratio of 40: 1. After incubation for 3 hr at 37°C assays are stopped by centrifugation and 51 Cr release from lysed cells are measured in a scintillation counter. Percentage of cellular cytotoxicity may be calculated as % maximal lysis which may be induced by adding 3% perchloric acid to target cells.
  • ADCP Antibody-dependent cellular phagocytosis
  • phagocytic cells such as macrophages or dendritic cells.
  • ADCP may be evaluated by using Tregs or MDSCs expressing CD38 as target cells engineered to express GFP or other labeled molecule.
  • Effctontarget cell ratio may be for example 4: 1. Effector cells may be incubated with target cells for 4 hours with or without anti-CD38 antibody. After incubation, cells may be detached using accutase.
  • Macrophages may be identified with anti-CD 1 lb and anti- CD14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescent in the CD1 1 D14 ⁇ macrophages using standard methods.
  • “Complement -dependent cytotoxicity” refers to a mechanism for inducing cell death in which an Fc effector domain of a target-bound antibody binds and activates complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.
  • complement receptors e.g., CR3
  • the ability of monoclonal antibodies to induce ADCC may be enhanced by engineering their oligosaccharide component.
  • Human IgGl or IgG3 are N-glycosylated at Asn297 with the majority of the glycans in the well-known biantennary GO, GOF, Gl, GIF, G2 or G2F forms.
  • Antibodies produced by non-engineered CHO cells typically have a glycan fucose content of about at least 85%. The removal of the core fucose from the biantennary complex-type oligosaccharides attached to the Fc regions enhances the ADCC of antibodies via improved FcyRllla binding without altering antigen binding or CDC activity.
  • niAbs may be achieved using different methods reported to lead to the successful expression of relatively high defucosylated antibodies bearing the biantennary complex-type of Fc oligosaccharides such as control of culture osmolality (Konno et ai, (2012) Cytotechnology 64:249-65), application of a variant CHO line Led 3 as the host cell line (Shields et al, (2002) J Biol Chem 277:26733-26740), application of a variant CHO line EB66 as the host cell line (Olivier et al, (2010) MAbs 2(4), Epub ahead of print; PMID:20562582), application of a rat hybridoma cell line YB2/0 as the host cell line (Shinkawa et al., (2003) J Biol Chem 278:3466-3473), introduction of small interfering RNA specifically against the a 1 ,6-fucosyltrasferase ( FUT8) gene (Mori e
  • ADCC elicited by anti-CD38 antibodies used in the methods of the invention may also be enhanced by certain substitutions in the antibody Fc.
  • Exemplary substitutions are for example substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EU index) as described in U.S. Pat. No.
  • the antibody that specifically binds CD38 comprises a substitution in the antibody Fc.
  • the antibody that specifically binds CD38 comprises a substitution in the antibody Fc at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EU index).
  • the antibody that specifically binds CD38 has a biantennary glycan structure with fucose content of about between 0% to about 15%, for example 15%, 14%, 13%, 12%, 11 % 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.
  • the antibody that specifically binds CD38 has a biantennary glycan structure with fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 1 1% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, l% or 0%
  • “Fucose content” means the amount of the fucose monosaccharide within the sugar chain at Asn297.
  • the relative amount of fucose is the percentage of fucose- contaming structures related to ail giycostractures. These may be characterized and quantified by multiple methods, for example: 1) using MALDI-TOF of N-glycosidase F treated sample (e.g. complex, hybrid and oligo- and high-mannose structures) as described in Intl. Pat. Pubi. No.
  • WO2008/077546 2) by enzymatic release of the Asn297 glycans with subsequent denvatization and detection/ quantitation by HPLC (UPLC) with fluorescence detection and/or HPLC-MS (UPLC-MS); 3) intact protein analysis of the native or reduced mAb, with or without treatment of the Asn297 glycans with Endo S or other enzyme that cleaves between the first and the second GlcNAc monosaccharides.
  • the oligosaccharides released may be labeled with a fluorophore, separated and identified by various complementary techniques which allow: fine characterization of the glycan structures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of the experimental masses with the theoretical masses, determination of the degree of sialylation by ion exchange HPLC (GiycoSep C), separation and quantification of the oligosacharride forms according to hydrophilicity criteria by normal-phase HPLC (GiycoSep N), and separation and quantification of the oligosaccharides by high performance capillary electrophoresis-laser induced fluorescence (HPCE-LIF).
  • MALDI matrix-assisted laser desorption ionization
  • ''Low fucose or "low fucose content” as used herein refers to antibodies with fucose content of about 0% - 15%.
  • Normal fucose or 'normal fucose content refers to antibodies with fucose content of about over 50%, typically about over 60%, 70%, 80% or over 85%.
  • the antibody that specifically binds CD38 may induce killing of Tregs, MDSCs and/or Bregs by apoptosis.
  • Methods for evaluating apoptosis are well known, and include for example annexin IV staining using standard methods.
  • the anti-CD38 antibodies used in the methods of the invention may induce apoptosis in about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of cells.
  • the Teffs or the immune suppressor cells reside in bone marrow or in peripheral blood.
  • the Teffs or the immune suppressor cells reside in bone marrow.
  • the Teffs or the immune suppressor cells reside in peripheral blood.
  • the antibody that specifically binds CD38 increases the ratio of CDS 1 T cells to Tregs.
  • the antibody that specifically binds CD38 increases the ratio of CD8 ⁇ central memory cells to CD8 " naive cells.
  • CD8 " central memory cells can be identified as CD45R07CD62L t sh cells.
  • CD8 + naive cells can be identified as CD45RO- /CD62L 4 cells.
  • the antibody that specifically binds CD38 is a non- agonistic antibody.
  • a non-agonistic antibody that specifically binds CDS 8 refers to an antibody winch upon binding to CD38 does not induce significant proliferation of a sample of peripheral blood mononuclear cells in vitro when compared to the proliferation induced by an isotype control antibody or medium alone.
  • the non-agonistic antibody that specifically binds CD38 induces proliferation of peripheral blood mononuclear cells (PBMCs) in a statistically insignificant manner.
  • PBMC proliferation may be assessed by isolating PBMCs from healthy donors and culturing the cells at 1 x 10 s cells/well in flat bottom 96-well plates in the presence or absence of a test antibody in 200 ⁇ RPMI After four day incubation at 37° C, 30 ⁇ ⁇ -thymidine (16.7 C ' i/mi ) may be added, and culture may be continued overnight.
  • Rhymidine incorporation may be assessed using a Packard Cobra gamma counter (Packard Instruments, Meriden, DT, USA), according to the manufacturer's instructions. Data may be calculated as the mean cpm ( ⁇ SEM) of PBMCs obtained from several donors. Statistical significance or insignificance between samples cultured in the presence or absence of the test antibody is calculated using standard methods.
  • DARZALEX 1 M (daratumumab).
  • DARZALEX iM (daratumumab) comprises a heavy chain variable region (VH) and a light chain variable region (VL) amino acid sequences shown in SEQ ID NO: 4 and 5, respectively, a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain complementarity determining region 1 (LCDRl), a LCDR2 and a LCDR3 of SEQ ID NOs: 9, 10 and 11 , respectively, and is of IgGl/ ⁇ subtype and described in U.S. Pat. No. 7,829,693.
  • DARZALEX ! ' yi (daratumumab) heavy chain amino acid sequence is shown in SEQ ID NO: 12 and light chain amino acid sequence shown in SEQ ID NO: 13.
  • the antibody that specifically binds CD38 competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody that specifically binds CD38 binds at least to region SKRNIQFSCKNJYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG IQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
  • SKRNIQFSCKNJYR SEQ ID NO: 2
  • EKVQTLEAWVIHGG IQ ID NO: 3 of human CD38 (SEQ ID NO: 1).
  • Antibodies may be evaluated for their competition with a reference antibody such as DARZALEXTM (daratumumab) having the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for binding to CD38 using well known in vitro methods.
  • a reference antibody such as DARZALEXTM (daratumumab) having the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for binding to CD38 using well known in vitro methods.
  • CHO cells recombinantly expressing CD38 may be incubated with an unlabeled reference antibody for 15 min at 4°C, followed by incubation with an excess of
  • test antibody fluorescently labeled test antibody for 45 min at 4°C. After washing in PBS/BSA, fluorescence may be measured by flow cytometry using standard methods. In another exemplar ' method, extracellular portion of human CD38 may be coated on the surface of an ELISA plate. Excess of the unlabeled reference antibody may be added for about 15 minutes and subsequently biotinylated test antibodies may be added. After washes in PBS/Tween, binding of the test biotinylated antibody may be detected using horseradish peroxidase (HRP)-conjugated streptavidin and the signal detected using standard methods. It is readily apparent that in the competition assays, the reference antibody may be labelled and the test antibody unlabeled.
  • HRP horseradish peroxidase
  • test antibody competes with the reference antibody when the reference antibody inhibits binding of the test antibody, or the test antibody inhibits binding of the reference antibody to CD38 by at least 80%, for example 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the epitope of the test antibody may further be defined for example by peptide mapping or hydrogen/deuterium protection assays using known methods, or by crystal structure determination.
  • Antibodies binding to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1) may be generated for example by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NOs: 2 and 3 using standard methods and those described herein, and characterizing the obtained antibodies for binding to the peptides using for example ELISA or mutagenesis studies.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof an anti-CD38 antibody that binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region
  • the epitope of the antibody used in the methods of the invention includes some or all of the residues having the sequences shown in SEQ ID NO: 2 or SEQ ID NO: 3.
  • the antibody epitope comprises at least one amino acid in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least one amino acid in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
  • the antibody epitope comprises at least two amino acids in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least two amino acids in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). In some embodiments, the antibody epitope comprises at least three ammo acids in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least three ammo acids in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
  • the antibody that specifically binds CD38 comprises the HCDR1, the HCDR2 and the HCDR3 amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively.
  • the antibody that specifically binds CDS 8 comprises the LCDR1, the LCDR2 and the LCDR3 ammo acid sequences of SEQ ID NOs: 9, 10 and 1 1, respectively.
  • the antibody that specifically binds CD38 comprises the HCDRI , the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 ammo acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10 and 1 1, respectively.
  • the antibody that specifically binds CD38 comprises the VH that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 and the VL that is 95%, 96%, 97%, 98% , 99% or 100% identical to SEQ ID NO: 5.
  • the antibody that specifically binds CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
  • the antibody that specifically binds CD38 comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13.
  • anti-CD38 antibodies that may be used in any embodiment of the invention are: mAb003 comprising the VH and the VL sequences of SEQ ID NOs: 14 and 15, respectively and described in U.S. Pat. No. 7,829,693. Trie VH and the VL of mAb003 may be expressed as IgGl/ ⁇ .
  • mAb024 comprising the VH and the VL sequences of SEQ ID NOs: 16 and 17, respectively, described in U.S. Pat. No. 7,829,693.
  • the VH and the VL of mAb024 may be expressed as IgGl/ ⁇ .
  • MOR-202 (MOR-03087) comprising the VH and the VL sequences of SEQ ID NOs: 18 and 19, respectively, described in US, Pat. No, 8,088,896.
  • the VH and the VL of MOR- 202 may be expressed as IgGl/' ⁇ .
  • Isatuximab comprising the VH and the VL sequences of SEQ ID NOs: 20 and 21, respectively, described in U.S. Pat. No. 8, 153,765.
  • the VH and the VL of isatuximab may be expressed as IgG l/ .
  • anti-CD38 antibodies that may be used in the methods of the invention include those described in Int. Pat. Publ. No. WO05/103083, Intl. Pat. Publ. No. WO06/125640, Intl. Pat. Publ, No. WO07/042309, Intl. Pat. Publ , No. WO08/047242 or Intl. Pat. Publ. No. WO 14/178820.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, compri sing administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, compri sing administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19 or a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21 or a time sufficient to treat the solid tumor.
  • the solid tumor is a melanoma.
  • the solid tumor is a lung cancer.
  • the solid tumor is a squamous non-small cell lung cancer (NSCLC).
  • NSCLC squamous non-small cell lung cancer
  • the solid tumor is a non-squamous NSCLC.
  • the solid tumor is a lung adenocarcinoma.
  • the solid tumor is a renal cell carcinoma (RCC) (e.g., a kidney clear cell carcinoma or a kidney papillary cell carcinoma), or a metastatic lesion thereof.
  • RCC renal cell carcinoma
  • the solid tumor is a mesothelioma.
  • the solid tumor is a nasopharyngeal carcinoma (NPC).
  • NPC nasopharyngeal carcinoma
  • the solid tumor is a colorectal cancer.
  • the solid tumor is a prostate cancer or castration-resistant prostate cancer.
  • the solid tumor is a stomach cancer.
  • the solid tumor is an ovarian cancer.
  • the solid tumor is a gastric cancer.
  • the solid tumor is a liver cancer.
  • the solid tumor is pancreatic cancer.
  • the solid tumor is a thyroid cancer.
  • the solid tumor is a squamous cell carcinoma of the head and neck.
  • the solid tumor is a carcinomas of the esophagus or gastrointestinal tract.
  • the solid tumor is a breast cancer.
  • the solid tumor is a fallopian tube cancer.
  • the solid tumor is a brain cancer.
  • the solid tumor is an urethral cancer.
  • the solid tumor is a genitourinary cancer.
  • the solid tumor is an endometriosis.
  • the solid tumor is a cervical cancer.
  • the solid tumor is a metastatic lesion of the cancer.
  • the solid tumor lacks detectable CD38 expression.
  • the solid tumor lacks detectable CDS 8 expression when CDS 8 expression in the solid tumor tissue or on cells isolated from the solid tumor is statistically insignificant when compared to a control, e.g. expression detected with anti-CD38 antibody vs expression detected with an isotype control antibody using well known methods.
  • Anti-CD38 antibodies used in the methods of the invention may also be selected de novo from, e.g., a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions nappik et at, (2000) JMol Biol 296:57- 86; Krebs et at, (2001) J Immunol Meth 254:67-84; Vaughan et al, ( 1996) Nature Biotechnology 14:309-314; Sheets et al, (1998) PITAS (USA) 95:6157-6162;
  • a phage display library where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions nappik et at, (2000) JMol Biol 296:57- 86; Krebs et at,
  • CD38 binding variable domains may be isolated from e.g., phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al, (2010) JMol Biol 397:385-96, and Intl. Pat. Publ. No. WO09/085462.
  • the antibody libraries may be screened for binding to human CD38 extracellular domain, the obtained positive clones further characterized, Fabs isolated from the clone lysates, and subsequentely cloned as full length antibodies.
  • Such phage display methods for isolating human antibodies are established in the art. See for example: U.S. Pat. No. 5,223,409, U.S. Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,427,908, U.S. Pat. No, 5,580,717, U.S. Pat. No. 5,969,108, U.S. Pat. No. 6,172, 197, U.S. Pat. No. 5,885,793, U.S. Pat. No. 6,521,404, U.S. Pat. No.
  • the anti-CD38 antibody is of IgGl , IgG2, IgG3 or IgG4 isotype.
  • the Fc portion of the antibody may mediate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody -dependent cellular phagocytosis (ADCP) or complement dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody -dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • Such function may be mediated by binding of an Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity or by binding of an Fc effector domain(s) to components of the complement system.
  • the effect(s) mediated by the Fc -binding cells or complement components result in inhibition and/or depletion of target cells, for example CD38-expressing cells.
  • Human IgG isotypes IgGl , TgG2, IgG3 and IgG4 exhibit differential capacity for effector functions.
  • ADCC may be mediated by IgGl and IgG3
  • ADCP
  • Antibodies that are substantially identical to the antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 may be used in the methods of the invention.
  • the term "substantially identical" as used herein means that the two antibody VH or VL amino acid sequences being compared are identical or have "insubstantial differences". Insubstantial differences are substitutions of I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in an antibody heavy chain or light chain that do not adversely affect antibody properties. Percent identity may be determined for example by pairwise alignment using the default settings of the AlignX module of Vector NT! v.9.0.0
  • the protein sequences of the present invention may be used as a query sequence to perform a search against public or patent databases to, for example, identify related sequences.
  • Exemplary programs used to perform such searches are the XBLAST or BLASTP programs (littp_/Avww_ncbi_nlm/nih_gov), or the GenomeQuest 13 ⁇ 41 (GenomeQuest, Westborough, MA) suite using the default settings.
  • Exemplary substitutions that may be made to the antibodies that specifically bind CDS 8 are for example conservative substitutions with an amino acid having similar charge,
  • any native residue in the VH or the VL may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis
  • Desired amino acid substitutions may be determined by those skilled in the art at the time such substitutions are desired. Amino acid substitutions may be done for example by PGR mutagenesis (U.S. Pat. No. 4,683,195).
  • variants may be generated using well known methods, for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Tip) and screening the libraries for variants with desired properties.
  • the generated variants may be tested for their binding to CD38, their ability to induce ADCC, ADCP or apoptosis, or modulate CDS 8 enzymatic activity in vitro using methods described herein.
  • the antibody that specifically binds CD38 may bind human CD38 with a range of affinities (K D ).
  • K D affinities
  • the antibody that specifically binds CD38 binds to CD38 with high affinity, for example, with a K D equal to or less than about 10 " ' M, such as but not limited to, 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) x lO "8 M, 10 "9 M, 10 "10 M, 10 "u M, 10 "S2 M, 10 “lj M, 10 "14 M, 10 “L M or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.
  • One exemplary affinity is equal to or less than 1 x10 s M
  • Another exemplary' affinity is equal to or less than 1x10 " M.
  • the antibody that specifically binds CDS 8 is a bispecific antibody.
  • the VL and/or the VH regions of the existing anti-CD38 antibodies or the VL and VH regions identified de novo as described herein may be engineered into bispecific full length antibodies.
  • Such bispecific antibodies may be made by modulating the CHS interactions between the monospecific antibody heavy chains to form bispecific antibodies using technologies such as those described in U.S. Pat, No, 7,695,936; Intl. Pat. Pub) . No, WO04/111233; U.S. Pat. Publ. No. US2010/0015133; U.S. Pat. Publ. No.
  • WO2011/143545 or U.S. Pat. Publ. No. US2012/0149876.
  • Additional bispecific stractures into which the VL and/or the VH regions of the antibodies of the invention may be incorporated are for example Dual Variable Domain Immunoglobulins (Inlt. Pat. Publ. No. WO2009/134776), or structures that include various dimerization domains to connect the two antibody arms with different specificity, such as leucine zipper or collagen dimerization domains (Int. Pat. Publ. No. WQ2012/02281 L U.S. Pat. No. 5,932,448; U.S. Pat, No. 6,833,441).
  • bispecific antibodies may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parental monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Intl.Pat. Publ, No.
  • the first monospecific bivalent antibody e.g., anti- CD38 antibody
  • the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange.
  • the incubation conditions may optimally be restored to non-reducing.
  • Exemplary reducing agents that may be used are 2- mercaptoethylamine (2- MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2- carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of: 2- mercaptoethylamine,
  • dithiothreitol and tris(2 ⁇ carboxyethyl)phosphine for example, incubation for at least 90 min at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.
  • Exemplary CH3 mutations that may be used in a first heavy chain and in a second heavy chain of the bispecific antibody are K409R and/or F405L.
  • the methods of the invention may be used to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.
  • the antibodies that specifically bind CDS 8 may be provided in the methods of the invention in suitable pharmaceutical compositions comprising the antibody that specifically bind CDS 8 and a pharmaceutically acceptable carrier.
  • the carrier may be diluent, adjuvant, excipient, or vehicle with which the antibodies that specifically bind CD38 are administered.
  • vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used . These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc.
  • concentration of the antibodies that specifically bind CD38 in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5%, usually to at least about 1% to as much as 15 or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21 sl Edition, Troy, D.B. eel., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.
  • the mode of administration of the antibodies that specifically bind CD38 in the methods of the invention may be any suitable route such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art.
  • the antibodies that specifically bind CDS 8 may be administered intratumorally, to a lymph node draining site for local delivery into the tumor using known methods.
  • the antibodies that specifically bind CD38 may be administered to a patient by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously or irrtraperitoneally.
  • i.v. infusion may be given over for example 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
  • the dose given to a patient is sufficient to alleviate or at least partially arrest the disease being treated ("therapeutically effective amount") and may be sometimes 0.005 rng to about 100 mg/kg, e.g. about 0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 1 8, 19, 20, 21 , 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.
  • a fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m 2 .
  • 1 and 8 doses e.g., 1 , 2, 3, 4, 5, 6, 7 or 8
  • 9 10, 1 1 , 12, 13, 14, 15, 16, 17, 1 8, 19, 20 or more doses may be given.
  • the administration of the antibodies that specifically bind CD38 in the methods of the invention may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration.
  • the repeated administration may be at the same dose or at a different dose.
  • the antibodies that specifically bind CD38 in the methods of the invention may be administered at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every four weeks by intravenous infusion.
  • the antibodies that specifically bind CD38 may be administered in the methods of the invention by maintenance therapy, such as, e.g. once a week for a period of 6 months or more.
  • the antibodies that specifically bind CD38 in the methods of the invention may be provided as a daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1 .0, 1 .1, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • the antibodies that specifically bind CDS 8 in the methods of the invention may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an e vent in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors.
  • the antibodies that specifically bind CD38 in the methods of the invention may be lyophiiized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.
  • the antibodies that specifically bind CD38 in the methods of the invention may be administered in combination with a second therapeutic agent.
  • the antibodies that specifically bind CD38 may be administered together with any one or more of the chemotherapeutic dmgs or other anticancer therapeutics known to those of skill in the art.
  • Chemotherapeutic agents are chemical compounds useful in the treatm ent of cancer and include growth inhibitory agents or other cytotoxic agents and include alkylating agents, anti-metabolites, anti- microtubule inhibitors, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors and the like.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and meth ylamel amines including altretamine, triethylenemelamine, trietylenephosphoramide,
  • alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®)
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as benzodopa, carboquone, meturedopa, and uredopa
  • ethylenimines and meth ylamel amines including altretamine, triethylenemel
  • triethylenethiophosphaoramide and trimethyiolomeiamine nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
  • sizofiran spirogermanium; tenuazonic acid; tnaziquone; 2,2',2"-trichlorotriethylamine: urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C " ); cyclophosphamide; thiotepa; members of taxoid or taxane family, such as pacliiaxel (TAXOL®docetaxel (TAXOTERE®) and analogues thereof; chlorambucil; gemcitabine; 6 "thioguanine; mercaptopurine; methotrexate;
  • platinum analogues such as cisplatin and earboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; no antrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
  • topoisomerase inhibitor RFS 2000; difiuoromethylomithine (DMFO); retinoic acid;
  • esperami cms; capecitabine inhibitors of receptor tyrosine kinases and/or angiogenesis, including NEXAVAR® (sorafemb), SUTENT® (sunitmib), VOTRIENTTM (pazopanib), PALLADIATM (toceranib), ZACTIMATM (vandeianib), RECENTTN® (cediranib), regorafemb (BAY 73-4506), axitinib (AG013736), lestaurtmib (CEP-701), TARCEVA® (erlotinib), IRESSATM (gefitinib), i ' iiloirn (afatinib), TYKERB® (lapatinib), neratinib, and the like, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti -estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-iniidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapnstone, and FARESTON® (toremifene); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
  • Exemplary agents that may be used in combination with the antibody that specifically binds CD38 in the methods of the invention include tyrosine kinase inhibitors and targeted anti -cancer therapies such as IRESSA iM (gefitinib) and Tarceva' 5, (erlotmib) and other antagonists of HER2, HER3, HER4 or VEGF,
  • Exemplar ⁇ ' HER2 antagonists include CP-724-714, HERCEPTINTM (trastuzumah), OMNTTARGTM (pertuzumab), TAK- 165, TYKERB® (lapatimb) (EGFR and HER2 inhibitor), and GW-282974.
  • Exemplary HER3 antagonists include anti-Her3 antibodies (see e.g., U.S. Pat. Publ. No.
  • HER4 antagonists include anti-HER4 siRNAs (see e.g., Maatta et al, Mol Biol Cell 17: 67-79, 2006,
  • An exemplar ' VEGF antagonist is
  • AvastinTM Be vacizumab
  • Exemplary agents that may be used in combination with the antibody that specifically binds CD38 in the methods of the invention include standard of care drags for solid tumors, or an immune checkpoint inhibitor.
  • the second therapeutic agent in the methods of the invention may be an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4 antibody.
  • the immune checkpoint inhibitor is an antagonistic anti- PD-1 antibody, an antagonistic anti-PD-Ll antibody, an antagonistic anti ⁇ PD-L2 antibody, an antagonistic anti-LAG3 antibody, or an antagonistic anti-TIM3 antibody.
  • the immune checkpoint inhibitor is an anti-PD-1 antibody.
  • the immune checkpoint inhibitor is an anti-PD-Ll antibody.
  • the immune checkpoint inhibitor is an anti-PD-L2 antibody.
  • the immune checkpoint inhibitor is an anti-LAG3 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-TIM3 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.
  • any antagonistic anti-PD-1 antibodies may be used in the methods of the invention.
  • Exemplary anti-PD-1 antibodies that may be used are OPVIDO® (nivolumab) and KEYTRUDA® (pembrolizumab).
  • OPVIDO® nivolumab
  • KEYTRUDA® pembrolizumab
  • OPVIDO® is described in for example US. Pat. No. 8,008,449 (antibody 5C4) and comprises the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25.
  • KEYTRUDA® pembrolizumab
  • U.S. Pat.No 8,354,509 comprises the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23.
  • nivolumab and pembrolizumab are also available through the CAS registry. Additional PD-1 antibodies that may be used are described in U.S. Pat. No. 7,332,582, U.S. Pat. Publ. No. 2014/0044738, Int. Pat. Publ, No. WO2014/17966 and U.S. Pat. Publ. No. 2014/0356363.
  • Antagonist refers to a molecule that, when bound to a cellular protein, suppresses at least one reaction or activity that is induced by a natural ligand of the protein.
  • a molecule is an antagonist when the at least one reaction or activity is suppressed by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than the at least one reaction or activity suppressed in the absence of the antagonist ⁇ e.g., negative control), or when the suppression is statistically significant when compared to the suppression in the absence of the antagonist.
  • Antagonist may be an antibody, a soluble ligand, a small molecule, a DNA or RNA such as siRNA.
  • a typical reaction or activity that is induced for example by PD-1 binding to its receptor PD- Li or PD-L2 may be reduced antigen-specific CD4 ⁇ or CD8 cell proliferation or reduced interferon- ⁇ (IFN- ⁇ ) production by T cells, resulting in suppression of immune responses against for example tumor.
  • a typical reaction or activity that is induced by TIM-3 binding to its receptor, such as galectin-9 may be reduced antigen specific CD4 + or CDS " cell proliferation, reduced IFN- ⁇ production by T cells, or reduced CD137 surface expression on CD4 ⁇ or CD8 " cells, resulting in suppression of immune responses against for example tumor.
  • an antagonistic PD-1 antibody specifically binding PD-1, an antagonistic PD-L2, an antagonistic antibody specifically binding TIM-3 induces immune responses by inhibiting the inhibitory pathways.
  • Anti-PD-Ll antibodies that enhance immune response may be used in the method of the invention (e.g. antagonistic anti-PD-LI antibodies).
  • Exemplary anti-PD-Ll antibodies that may be used are durvalumab, atezolizumab and aveiumab, and those described in, for example, U.S. Pat. Pub!. No. 2009/0055944, U.S. Pat. No. U.S. Pat, No, 8,552, 154, U.S. Pat. No. 8,217, 149 and U.S. Pat. No. 8,779,108.
  • Durvalumab comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27
  • Atezolizumab comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:
  • Aveiumab comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25 for a time sufficient to enhance the immune response.
  • the invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD 38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23 for a time sufficient to enhance the immune response.
  • the invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 for a time sufficient to enhance the immune response.
  • the invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29 for a time sufficient to enhance the immune response.
  • the invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD 38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31 for a time sufficient to enhance the immune response.
  • the invention also provides for a method of treating a patient having a colorectal cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-1 antibody for a time sufficient to treat the colorectal cancer.
  • the invention also provides for a method of treating a patient having a colorectal cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-Ll antibody for a time sufficient to treat the colorectal cancer.
  • the invention also provides for a method of treating a patient having a colorectal cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an an tagonistic anti-PD-L2 antibody for a time sufficient to treat the colorectal cancer.
  • the invention also provides for a method of treating a patient having a lung cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-1 antibody for a time sufficient to treat the lung cancer.
  • the invention also provides for a method of treating a patient having a lung cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-L 1 antibody for a time sufficient to treat the lung cancer.
  • the invention also provides for a method of treating a patient having a lung cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-L2 antibody for a time sufficient to treat the lung cancer.
  • the invention also provides for a method of treating a patient having a prostate cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-1 antibody for a time sufficient to treat the prostate cancer.
  • the invention also provides for a method of treating a patient having a prostate cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD 38 in combination with an antagonistic anti-PD-Ll antibody for a time sufficient to treat the prostate cancer.
  • the invention also provides for a method of treating a patient having a prostate cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 in combination with an antagonistic anti-PD-L2 antibody for a time sufficient to treat the prostate cancer.
  • Anti-LAG-3 antibodies that enhance immune response may be used in the methods if the invention.
  • Exemplary anti-LAG-3 antibodies that may be used are those described in, for example, Int. Pat. Pubi. No. WO2010/019570.
  • Anti-CTLA-4 antibodies that enhance immune response may be used in the methods if the invention.
  • An exemplary anti-CTLA-4 antibody that may be used is ipilimumab.
  • Anti-PD-1, anti-PD-Ll, anti-PD-L2, anti-LAG3, anti-TlM3 and anti-CTLA-4 antibodies that may be used in the methods of the invention may also be generated de novo using methods described herein.
  • anti-PDl antibodies comprising the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33 may be used.
  • anti-PDl antibodies comprising the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 35 may be used.
  • anti-TIM-3 antibodies comprising the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37 may be used.
  • anti-TIM-3 antibodies comprising the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39 may be used.
  • E1VMTQS1 1 SVSPGERATLSCRASQSLSDYLH ⁇ 3 ⁇ 4YQQKPGQAPRL,L1KSASQS1SG IPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNGHSFPYTFGQGTKLEIK
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-l antibody comprising the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 35 for a time sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti ⁇ TIM-3 antibody comprising the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37 for a rime sufficient to treat the solid tumor.
  • the invention also provides for a method of treating a patient having a solid tumor, compri sing administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-TIM-3 antibody comprising the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39 for a time sufficient to treat the solid tumor.
  • the combination of the antibody that specifically binds CD38 and the second therapeutic agent may be administered over any convenient timeframe.
  • the antibody that specifically binds CD38 and the second therapeutic agent may be administered to a patient on the same day, and even in the same intravenous infusion.
  • the antibody that specifically binds CD38 and the second therapeutic agent may also be administered on alternating days or alternating weeks or months, and so on.
  • the antibody that specifically binds CD38 and the second therapeutic agent may be administered with sufficient proximity in time that they are simultaneously present (e.g., in the serum) at detectable levels in the patient being treated.
  • an entire course of treatment with the antibody that specifically binds CD38 consisting of a number of doses over a time period is followed or preceded by a course of treatment with the second therapeutic agent, consisting of a number of doses.
  • a recovery period of 1 , 2 or several days or weeks may be used between administration of the antibody that specifically binds CD38 and the second therapeutic agent.
  • the antibody that specifically binds CD38 or a combination of the antibody that specifically binds CD38 and the second therapeutic agent may be administered together with any form of radiation therapy including external beam radiation, intensity modulated radiation therapy (IMRT), focused radiation, and any form of radiosurgery including Gamma Knife, Cyberknife, Linac, and interstitial radiation (e.g. implanted radioactive seeds, GliaSite balloon), and/or with surgery.
  • IMRT intensity modulated radiation therapy
  • radiosurgery including Gamma Knife, Cyberknife, Linac, and interstitial radiation (e.g. implanted radioactive seeds, GliaSite balloon), and/or with surgery.
  • stereotactic radiosurgery involves the precise delivery of radiation to a tumorous tissue, for example, a brain tumor, while avoiding the surrounding non- tumorous, normal tissue.
  • the dosage of radiation applied using stereotactic radiosurgery may vary, typically from 1 Gy to about 30 Gy, and may encompass intermediate ranges including, for example, from 1 to 5, 10, 15, 20, 25, up to 30 Gy in dose. Because of noninvasive fixation devices, stereotactic radiation need not be delivered in a single treatment.
  • the treatment plan may be reliably duplicated day-to-day, thereby allowing multiple fractionated doses of radiation to be delivered.
  • the radiosurgery When used to treat a tumor over time, the radiosurgery is referred to as "fractionated stereotactic radiosurgery" or FSR.
  • stereotactic radiosurgery refers to a one-session treatment. Fractionated stereotactic radiosurgery may result in a high therapeutic ratio, i.e., a high rate of killing of tumor cells and a low effect on normal tissue.
  • the tumor and the normal tissue respond differently to high single doses of radiation vs. multiple smaller doses of radiation. Single large doses of radiation may kill more normal tissue than several smaller doses of radiation may.
  • IMRT Intensity-modulated radiation therapy
  • 3DCRT three-dimensional conformal radiation therapy
  • IMRT allows the radiation dose to confonn more precisely to the three-dimensional (3-D) shape of the tumor by modulating the intensity of the radiation beam in multiple small volumes. Accordingly, IMRT allows higher radiation doses to be focused to regions within the tumor while minimizing the dose to surrounding normal critical structures. IMRT improves the ability to confonn the treatment volume to concave tumor shapes, for example, when die tumor is wrapped around a vulnerable structure, such as the spinal cord or a major organ or blood vessel.
  • Subcutaneous administration of pharmaceutical compositions comprising an antibody that specifically binds CD38 and a hyaluronidase
  • the antibody that specifically binds CD38 may be administered as a pharmaceutical composition comprising the antibody that specifically binds CD38 and a hyaluronidase subcutaneo usly .
  • the concentration of the antibody that specifically binds CD38 in the pharmaceutical composition administered subcutaneous! ⁇ ' may be about 20 mg/ml.
  • the pharmaceutical composition administered subcutaneously may comprise between about 1,200 mg - 1,800 mg of the antibody that specifically binds CD38.
  • the pharmaceutical composition administered subcutaneously may comprise about 1,200 mg of the antibody that specifically binds CD38.
  • the pharmaceutical composition administered subcutaneously may comprise about 1,600 mg of the antibody that specifically binds CD38.
  • the pharmaceutical composition administered subcutaneously may comprise about 1,800 mg of the antibody that specifically binds CD38.
  • the pharmaceutical composition administered subcutaneously may comprise between about 30,000 U - 45,000 U of the hyaluronidase.
  • the pharmaceutical composition administered subcutaneously may comprise about 1,200 mg of the antibody that specifically binds CD38 and about 30,000 U of the hyaluronidase.
  • the pharmaceutical composition administered subcutaneously may comprise about 1,800 mg of the antibody that specifically binds CD38 and about 45,000 U of the hyaluronidase.
  • the pharmaceutical composition administered subcutaneously may comprise about 1,600 rng of the antibody that specifically binds CD38 and about 30,000 U of the hyaluronidase.
  • the pharmaceutical composition administered subcutaneously may comprise about 1 ,600 mg of the antibody that specifically binds CD38 and about 45,000 U of the hyaluronidase.
  • the pharmaceutical composition administered subcutaneously may comprise the hyaluronidase rHuPH20 having the amino acid sequence of SEQ ID NO: 40.
  • rHuPH20 is a recombinant hyaluronidase (HYLENEX® recombinant) and is described in Int. Pat. Publ. No. WO2004/078140.
  • Hyaluronidase is an enzyme that degrades hyaluronic acid (EC 3 ,2.1.35) and lowers the viscosity of hyaluronan in the extracellular matrix, thereby increasing tissue permeability.
  • the administration of the pharmaceutical composition comprising the antibody that specifically binds CD38 and the hyaluronidase may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.
  • the pharmaceutical composition comprising the antibody that specifically binds CD38 and the hyaluronidase may be administered once weekly for eight weeks, followed by once in two weeks for 16 weeks, followed by once in four weeks.
  • the pharmaceutical compositions to be administered may comprise about 1,200 mg of the antibody that specifically binds CD38 and about 30,000 U of hyaluronidase, wherein the concentration of the antibody that specifically binds CD38 in the pharmaceutical composition is about 20 mg/'ml.
  • the pharmaceutical compositions to be administered may comprise about 1,800 mg of the antibody that specifically binds CD38 and about 45,000 U of hyaluronidase.
  • the pharmaceutical compositions to be administered may comprise about 1,600 mg of the antibody that specifically binds CD38 and about 30,000 U of hyaluronidase.
  • the pharmaceutical compositions to be administered may comprise about 1,600 mg of the antibody that specifically binds CD38 and about 45,000 U of
  • the pharmaceutical composition comprising the antibody that specifically binds CDS 8 and the hyaluronidase may be administered subcutaneously to the abdominal region.
  • the pharmaceutical composition comprising the antibody that specifically binds CD38 and the hyaluronidase may be administered in a total volume of about 80 ml, 90 ml, 100 ml, 1 10 ml or 120 ml .
  • 20 mg/ml of the antibody that specifically binds CDS 8 in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, 0.04% poiysorbate 20, pH 5.5 may be mixed with rHuPH20, 1.0 mg/mL (75-150 kU/mL) in 10 mM L-Histidine, 130 mM NaCl, 10 mM L-Met ionine, 0.02% Poiysorbate 80, pH 6.5 prior to
  • An antibody that specifically binds CDS 8 for use in treating a patient having a solid tumor 1.
  • An antibody that specifically binds CD38 for use in treating a patient having a regulatory T cell (Treg) mediated disease is provided.
  • An antibody that specifically binds CD38 for use in treating a patient having a myeloid-derived suppressor cell (MDSC) mediated disease, comprising administering to the subject a therapeutically effective amount of an antibody that specifically binds CD38.
  • MDSC myeloid-derived suppressor cell
  • T regulatory T cell comprising contacting the T regulatory cell with an antibody that specifically binds CD38.
  • MDSC myeloid-derived suppressor cell
  • An antibody that specifically binds CD38 for use in treating a patient having a solid tumor, comprising reducing the number of regulatory T cells in a patient by administering the patient an antibody that specifically binds CDS 8.
  • An antibody that specifically binds CD38 for use in treating a patient having a solid tumor comprising reducing the number of myeloid-derived suppressor cells (MDSC) cells in a patient by administering the patient an antibody that specifically binds CD38.
  • MDSC myeloid-derived suppressor cells
  • Tregs regulatory T cells
  • MDSC myeloid-derived suppressor cells
  • ADCC antibody-dependent cell cytotoxicity
  • ADCP antibody-dependent ceil phagocytosis
  • CDC cell-dependent cytotoxicity
  • CD38 The antibody that specifically binds CD 38 for use according to embodiment 21, wherein the CD1 lb3 ⁇ 4LA-DROD14 " CD33 + CD15 + cells express CD38.
  • ADCC antibody-dependent cell cytotoxicity
  • ADCP antibody-dependent cell phagocytosis
  • CDC complement-dependent cytotoxicity
  • VH heavy chain variable region
  • VL light chain variable region
  • SKRNIQFSCKNIYR SEQ ID NO: 2
  • EKV QTLE A WV IHGG SEQ ID NO: 3 of human CD38
  • HCDR complementarity determining regions 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively.
  • LCDR complementarity determining regions
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor, wherein the antibody is administered in combination with the anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23.
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor wherein the antibody is administered in combination with the anti-PD-1 antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25.
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in enhancing an immune response in a patient.
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in enhancing an immune response in a patient wherein the antibody is administered in combination with the anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23.
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27.
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VI, of SEQ ID NO: 5 for use in treating a patient having a solid tumor wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29.
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31.
  • An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VI, of SEQ ID NO: 5 for use in enhancing an immune response in a patient wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27.
  • PBMCs Peripheral blood mononuclear cells
  • cell lineage panel PerCPCy5.5 -CD19 (cloneHIB 19; Becton Dickinson [BD]), APCa-CD24 (SN3; eBiosaence), PC7a-CD3 (UCHT-1; Beckman Coulter), V500a-CD16 (3G8; BD), and PEa-CD56 (MY; BD); regulatory T cell (T Jeg ) panel: APCa-CD25 (2A3; BD), PEa- CD127 (HIL-7R-M21 ; BD), APC-H7a-HLA-DR (G46-6; BD), and PerCPa-CD4 (L200; BD); na
  • CD38 expression was evaluated using Alexa 647 labeled antibody mAb 003 described in U.S. Pat. No. 7,829,693 having the VH and the VL sequences of SEQ ID NO: 14 and SEQ ID NO: 15.
  • the blood samples were prepared using different Lyse-wash methods.
  • For bone marrow aspirate samples either membrane or intracellular staining was performed with various antibodies.
  • Becton Dickinson FACSLysing solution was used for lysing red blood cells in peripheral blood samples and Fix and Perm cell permeabilization reagents from Invitrogen were used for intracellular staining of bone marrow aspirate samples. Stained samples were acquired on FACS Canto II flow cytometers and data was analyzed using FacsDiva software. Absolute counts of immune cell populations in the blood samples and as percent of lymphocytes in bone marrow samples were determined at all the time points tested.
  • TCR T-cel! receptor
  • T-cell diversity was analyzed by deep sequencing of TCR rearrangements to assess CD8 + T-cell clonality using genomic DNA from PBMC samples.
  • TCR sequencing was performed using Adaptive Biotechnologies commercial Immunoseq lM assay, and analysis was performed using prequalified multiplex polymerase chain reaction (PGR) assays (TR2015CRO-V-01 ), which were composed of forward and reverse primers that directly targeted the family of variable (V) genes (forward primers) and joining (J) genes (reverse primers).
  • V variable
  • J joining
  • each amplicon was amplified a second time with forward and reverse primers containing the universal sequence and adaptor sequence needed for DNA sequencing by lllumina.
  • PBMCs were seeded on 96 well plates (2 ⁇ 10 5 cells/well) and stimulated for 5 days with a cocktail of 23 major histocompatibility complex (MHC) class I-restricted viral peptides from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus (2 g/ml; CEF peptide pool ; PANATecs*) or an equivalent number of 25- Gy irradiated allogeneic PBMCs from healthy donors. Unstimulated PBMCs and PBMCs stimulated with anti-CD3/CD28-coated beads served as negative and positive controls, respectively.
  • MHC major histocompatibility complex
  • interferon ⁇ IFN- ⁇
  • ELISA sandwich enzyme-linked immunosorbent assay
  • CFSE Carboxyfluorescein succinimidyl
  • PBMCs from healthy donors were labelled with PerCP-Cy5.5a-CD3 (SK7; BD), KOa-CD45, (J33; Beckman Coulter), V450a-CD4 (SK3; BD), PEa-CD25 (M-A251, BD), PE Cy7a-CD127 (HIL-7R-M21 ; BD), and APCa-CD38 ( ⁇ -7; BD) and sorted by FACS Aria (BD).
  • Sorted effector cells were labelled with carboxyfluorescein succinimidyl ester (CFSE; eBioscience) and stimulated with anti-CD3/CD28-coated beads in the presence or absence of CD38 + Tregs or CD38 " Tregs (1: 1 Treg to effector cell ratio) in RPMI plus 10% fetal calf serum . After 72 hours, flow cytometry was performed and the percent dilution of CFSE was used as a surrogate for T-cell proliferation.
  • CFSE carboxyfluorescein succinimidyl ester
  • MDSC Myeloid derived suppressor cell
  • PBMC from three normal healthy donors were co-cultured with myeloma tumor cell lines (RPMI8226, U266, H929) for six days, and evaluated for the production of granulocytic MDSC (G-MDSC) (CDl lb + CD14 " HLA " DR:CD15 + CD33 + ) as described in Gorgun et al. Blood 121:2975-87, 2013.
  • G-MDSC granulocytic MDSC
  • G-MDSC granulocytic MDSC
  • Gating strategy for flow cytometric evaluation of G-MDSC included CD l lb + as the first gate, followed by CD 14 " and HLA DR gating, and then followed by CD15 + and CD33 ⁇ gating.
  • G-MDSCs were cell sorted and evaluated for CD38 expression levels and sensitivity to DARZALEX IM (daratumumab) mediated ADCC. To evaluate the effect of DARZALEXTM
  • PBMCs peripheral blood mononuclear cells
  • RPMI phosphate-buffered saline
  • responders are defined as subjects with a Best Response per IRC of sCR, VGPR and PR, and non-responders are defined as subjects with a Best Response per IRC of MR, SD and PD
  • Different statistical comparisons included (i) baseline levels between responders and non-responders, (ii) baseline versus on treatment for responders and for non- responders, (iii) percent changes between responders and non-responders, (iv) ratio changes of baseline versus on treatment.
  • Each comparison included first a test for normality with a Shapiro-Wilk test (Royston (1995) Remark AS R94: A remark on Algorithm AS 181 : The W test for normality. Applied Statistics, 44, 547-551). Almost exclusively, the data was found to not have a normal distribution.
  • the differential level testing included conducting both a non-parametric Wilcox rank sum test (Hollander and Wolfe (1973), Nonparametric Statistical Methods.
  • the target population for Study 54767414MMY2002 is patients with advanced multiple myeloma who received at least 3 prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD) or double refractory to a PI and an IMiD.
  • PI proteasome inhibitor
  • IMD immunomodulatory drug
  • OR R overall response rate
  • TTP time to progression
  • PFS progression free survival
  • OS overall survival
  • Group A DARZALEX 1M (daratumumab) 16 mg/kg: Cycles 1 and 2: Days 1, 8, 15, and 22 (weekly), Cycle 3 to 6: Days 1 and 15 (every other week), Cycles 7+: Day 1 (every 4 weeks). Each cycle was 4 weeks.
  • Group B DARZALEX lM (daratumumab) 8 mg/kg: Cycle 1+: Day 1 (every 4 weeks).
  • the primary objective of the study was to determine the efficacy of 2 treatment regimens of DARZALE lM (daratumumab), as measured by the OR (CR + PR), in subjects with multiple myeloma who have received at least 3 prior lines of therapy including a PI and an IMiD or whose disease is double refractory to both a PI and an IMiD (Clinical Study Report: An Open-label, Multicenter, Phase 2 Trial Investigating the Efficacy and Safety of DARZ LEX lM (daratumumab) in Subjects With Multiple Myeloma Who Have Received at Least 3 Prior Lines of Therapy (Including a Proteasome Inhibitor and IMiD) or are Double Refractor ' to a Proteasome Inhibitor and an IMiD. EDMS-ERI-92399922).
  • the secondary objectives of this study included evaluation of the safety and to.lerabi.lity of D ARZALEXTM (daratumurnab) demonstration of additional measures of efficacy (e.g, clinical benefit, TTP, PFS, and OS) along with assessment of
  • Stage 1 of Part 1 I subject (6%) responded in the 8 mg/kg group, and 5 subjects (31%) responded in the 16 mg/kg group. Therefore, only the 16 mg kg group was expanded in Stage 2 of Part 1 and in Part 2.
  • CD38 is expressed on a variety of immune and hematopoietic cells. Broad immune profiling by flow cytometry was performed to examine the effect of
  • D ARZALEXTM (daratumurnab) on immune cell subsets and the association of baseline levels of these cells to clinical response.
  • Various cell populations including T-cells (CD3 + , CDC, CD8 + and regulatory T-cells (Treg)), B-cells (CD 19 " ), NK cells, monocytes (CD14 + ), leukocytes, and neutrophils were e valuated by flow cytometry in peripheral blood and bone marrow aspirates at baseline and following DARZALEX lM
  • Lymphocytes Leukocytes, Monocytes and Neutrophils
  • NK cells CD 16 + CD56 i
  • activated NK cells CD 16 + CD56 dus
  • DARZALEXTM daratumumab
  • Lymphocytes were noted to increase with DARZALEX 1J l (daratumumab) treatment (Figure 1) even though B ceils showed only a minimal increase (see above).
  • various T-ce!l populations were studied (CD3 CD4 + , CD8 + T cells, regulator ' T cells) in both peripheral blood and bone marrow.
  • CD3 " , CD4 + and CD8 + T-cells were increased in peripheral blood (both absolute counts/ ⁇ and percentage of lymphocytes) following DARZALEX 1 M (daratumumab) treatment.
  • Figure 2 shows the percent change of absolute counts of CD3 1 T-cells (CD45 + CD3 ⁇ ) from baseline in peripheral blood over time for every- patient.
  • the black line in the Figure shows the median absolute counts x 10 e cells ⁇ L for all patients. Only visits with more than 2 observations were included into the Figure.
  • Figure 3 shows the % change of absolute counts of CD4 + T-cells (CD45 + CD3 + CD4 + ) from baseline in peripheral blood over time for every patient.
  • the black line in the Figure shows the median for all patients.
  • Figure 4 shows the % change of absolute counts of CD8 + T-cells (CD45 f CD3 + CD8 ) from baseline in peripheral blood over time for every patient. The black line in the Figure shows the median for all patients. Only visits with more than 2 obsen/ations were included into the Figure.
  • Table 2 shows the Wilcoxon signed-rank test results for the comparison of each T-cell subpopulation in peripheral blood between responders and non-responders for percent change of absolute counts to baseline.
  • total T-cells CD45 + CD3 ⁇ as a percentage of lymphocytes
  • CD8 + T-cells CD45XD3XD8 + as a percentage of lymphocytes
  • Table 3 shows the iicoxon signed-rank test results for the various T ceils as % lymphocytes in bone marrow?.
  • Figure 5 shows the percentage (%) of CD45 r CD3 + cells over time during DARZALEX lM (daratumumab) treatment (both responders and non-responders included in the graph).
  • Figure 6 shows the % CD45 + CD3 "! CD8 + cells over time during DARZALEX lM (daratumumab) treatment (both responders and non-responders included in the graph).
  • Figure 6 shows the % CD45 + CD3 "! CD8 + cells over time during
  • DARZALEX J M (daratumumab) treatment (both responders and non-responders included in the graph).
  • Treg cells were identified as the CD3 + CD4 + CD25 " CD127 dim cell population in a sample.
  • the ratio of CDS " T cells to Tregs was assessed in the peripheral blood and bone marrow in patients treated with DARZALEX 1M (daratumumab) over time. The ratio increased in both the periphery and bone marrow.
  • Figure 7 A shows the median values of the CD8 + /Treg and CD8 + CD4 T cell ratios of all patients per time point in peripheral blood.
  • Figure 7B shows the median values of the CD8 + /Treg and CD87CD4 " T-cell ratios of all patients per time point in bone marrow.
  • the changes in the ratios of absolute counts of CD8 + Tregs and CD8 CD4 + were significant in peripheral blood over time of treatment (Table 6) and in bone marrow (Table 7), Wilcoxon signed-rank test.
  • Study GEN501 was the first-in-human clinical study of DARZALEXTM (daratumumab) in subjects with MM. It is a Phase 1/2, dose-escalation, safety study divided into 2 parts. Part 1 is an open-label, dose-escalation study; Part 2 is an open-label, single-arm study with multiple cohorts, based on the dose levels established in Part 1
  • Part 1 10 dose levels of DARZALEX 1 (daratumumab) were evaluated: 0.005, 0.05, 0.10, 0.50, 1, 2, 4, 8, 16, and 24 mg/kg. The 2 lowest dose cohorts were allocated 1 (+3) subject(s) each, and a standard 3 (+3) subject allocation was applied to the remaining 8 dose cohorts. Part 2 was an open-label, single study including two dose levels, 8 mg/kg and 16 mg/kg. Part 1 included 32 subjects and Part 2 included 72 subjects.
  • Example 5 DARZALEX 1 M (daratumumab) treatment induces T cell clonality in patients
  • TCR T-ceil receptor
  • Figure 8B shows the fold change in clonalitv in individual patients. Responders are marked with the star. This data suggests that the T cell expansion noted with
  • DARZALEXTM (daratumumab) treatment may be clonal in nature.
  • FIG. 8C shows the % CIA for individual patients.
  • Group A: responders, Group B: non-responders. Statistically significant difference was observed between responders and non-responders (p 0.037).
  • Figure 81) shows the sum of absolute change in abundance (CIA) in responders and non- responders for each expanded T cell clone.
  • Figure 8F shows the maximum CIA of a single T-cell clone in responders (Group A) and non-responders (Group B).
  • CIA was obtained by identifying significant differences in clonal abundance between two samples using Fisher's exact test (DeWitt et al. J. Virol. 2015) and summing the absolute change in abundance for each expanded clone.
  • lymphocytes were increased in both peripheral blood and bone marrow during DARZALEX lM (daratumumab) treatment. This increment was attributed to increased numbers of both CD4 + and CDS 4 cells.
  • CD8 ⁇ T-cell phenotype was studied in patients treated with DARZALEX lM (daratumumab) over time in a subset of 17 patients enrolled in the GEN 501 study.
  • CDS f cells from patients were identified as naive (CD45R07CD62L + ) (T N ) or central memory (TC M ) (CD45R07CD62L +Wsh ) cells using standard protocols.
  • Figure 9A shows the % of CD8 + nai e cells (% of CD8 T cells) and Figure 9B shows the % of CDS + central memory cells.
  • White squares indicate patients that achieved at least minimal response (> MR.) and black squares indicate patients that had stable disease or progressive disease. A significantly greater decrease in CD8 + naive T cells was apparent in patients who responded to treatment (data not shown).
  • Figure 9C shows that
  • DARZALEXTM (daratumumab) treatment increased the percentage in HLA Class I- restricted T cells, which partially drive the virus-specific and alloreactive T cell responses
  • Figure 9D shows that the expanding effector memory T cells expressed low levels of CDS 8. It is important to note that these T cells display normal and even increased functional activity against viral peptides and alloantigens (see Example 8). From these functional results we concluded that there is an expansion of, or improved activity of, antigen-experienced T cells against viral and alloantigens during DARZALEX IM (daratumumab) treatment. These data suggest that, unlike regulatory cell subsets, effector T cells do not need CD38 expression to properly function and expand.
  • Tregs Regulatory T-cells
  • FIG. 10A A subpopulation of peripheral Tregs (10% ⁇ 10%) expressed high levels of CD38 prior to Treg activation.
  • Figure 10A top panel shows the frequency of the Tregs in the CD3 + CD4 + cell population (P4 cell population) at baseline.
  • Figure 10A bottom panel shows the subset of Tregs expressing high CD38 (PS cell population).
  • FIG. 10B The frequency of Tregs after DARZALEXTM (daratumumab) treatment is shown in Figure 10B, top panel (P4 cell population).
  • Figure 10B, bottom panel shows that the CD38 hig!l Tregs (P5 cells) was the most significantly depleted Treg population after 1 st DARZALEXTM (daratumurnab) infusion.
  • Figure IOC shows the % of CD38 hl8h Tregs from total CD3 " cells at baseline, week 1 , week 4, week 8, relapse, and 6 months after the end of treatment (EOT).
  • CD38 + Tregs with DARZALEX lM (daratumurnab) treatment
  • the suppressive capacity of CD38 f Tregs versus CD38 ⁇ Tregs on autologous CD3 1 T cells was assessed.
  • CD38 + Tregs suppressed T-cell proliferation more robustly (9.9% cell proliferation observed) than CD38 " Tregs (53.2% cell proliferation observed) or the negative control (74.9% cell proliferation observed) ( Figure 10E).
  • CD38 " granulocytic MDSCs (CDl lb + CD14 " HLA-DR " CD15 + CD33 + ) were generated in vitro from PBMCs isolated from patients at baseline and from patients who had received one infusion of DARZALEX lM (daratumurnab).
  • Figure 11 shows the flow cytometry histogram of identified MDSCs ( Figure 11, top histogram, boxed cell population). Approximately half of the MDSCs expressed CD38 ( Figure 11, middle graph; circled P7 cell population). The CD38 hlgb MDSCs were nearly depleted in patients treated with DARZALEX 1 M (daratumurnab) ( Figure 11, bottom graph; circled P7 cell population).
  • the CD38 high lineage nonspecific MDSCs were depleted with DARZALEXTM (daratumurnab) treatment over time in both non-responders and patients who have at least Minimal Repose to treatment.
  • Figure 12 shows that the percentage of the CD38 iuga MDSCs was reduced to nearly 0% in patients at 1 w eek.. 4 weeks or 8 weeks of treatment.
  • the CD38 mgh lineage nonspecific MDSCs returned to baseline after the end of treatment.
  • FIG. 13 shows that the patients 2, 4, 15, 16 and 17 having the highest percentage of CD38 hlgh MDSC (as shown in Figure 1 1) and classified as patients with P or MR, had a Progression-Free Survival (PFS) of at least 8 months.
  • the CD38 high lineage nonspecific MDSCs were also sensitive to DARZALEXTM (daratumumab)-induced ADCC in vitro.
  • ADCC assays were performed using CD38 Msa MDSCs from two donors and Daudi cells as control target cells with effectontarget cell ratio of 50: 1.
  • Figure 14 shows the results of the experiment from one donor.
  • DARZALEX lM (daratumumab) induced lysis of MDSC cells.
  • DARZALEX lM (daratumumab)-induced changes in T-cell populations and clonaiity.
  • the percentage of MDSCs was between about 10%-37% and between about 10%- 27% of PBMCs in the analyzed samples from, the NSCLC and prostate cancer patients, respectively.
  • CD38 expression was identified in 80-100% of Lin " CD14 HLADR "flow MDSCs from PBMCs from NSCLC patients and in 70-100% of MDSCs from PBMCs from prostate cancer patients.
  • DARZALEX llvl (daratumumab)
  • FIG. 16A shows the anti-viral response of one representative patient with VGPR.
  • Figure 16B shows the anti-viral response of one representative patient with CR
  • Figure 16C shows the anti-viral response of one representative patient with PD.
  • Figure 16D shows the anti-viral response of one representative patient with MR.
  • error bars represent standard error of the mean of duplicate cultures.
  • NK ceils regulatory T-cells (Tregs), regulatory B-ceils (Bregs), and myeloid derived suppressor cells (MDSCs)
  • DARZALEX lM daratumumab
  • Figure 17A shows a histogram of expression of CDS 8 in immune cells from a healthy donor
  • Figure 17B shows a histogram of expression of CD38 in immune cells from a multiple myeloma patient.
  • CDS 8 expression was highest on NK cells, followed by monocytes, B and T cells.
  • CDS 8 expression was highest on plasma cells, followed by a subset of B cells, NK cells, monocytes, B -cells and T-cells.
  • Figure 17C shows a comparison of the mean fluorescent intensity (MFI) of CD38 across NK cells, Tregs, Bregs, B- and T-cells cells from relapsed and refractory myeloma patients, demonstrating that after plasma ceils, NK cells expressed the highest levels of CDS 8, followed by regulatory T-cells (Tregs) and regulatory B-cells (Bregs).
  • MFI mean fluorescent intensity
  • CIPs complement inhibitory proteins
  • CD59 NK cells express very low levels of CD59 and CD55, while other T and B cell populations express much higher levels. This could also contribute to the variability of DARZALEX lM (daratumumab) sensitivity across immune cell subtypes (data not shown). Discussion
  • DARZALEXTM (daratumumab) through reduction of CD38 + immune suppressive cellular populations and concomitant induction of helper and cytotoxic T-cell expansion, production of IFN- ⁇ in response to viral peptides, and increased TCR clonality, indicating an improved adaptive immune response.
  • MDSCs and Bregs express CD38 and were susceptible to DARZALEXTM (daratumumab) treatment. These cells are known to be present in the tumor niicroenvironment and contribute to tumor growth, immune evasion, angiogenesis, metastasis, and production of suppressive cytokines. In addition to these CD38 + suppressive cellular subsets, a novel subpopulation of regulator ⁇ ' T cells
  • CD38 1 immune-regulatory cells may reduce local immune suppression within the myeloma niicroenvironment and allow positive immune effector cells to expand and contribute to antitumor response.
  • T-cell repertoire was examined in a subset of patients.
  • the skew in T-cell clonality was greater in patients with a good clinical response, and was correlated with the increase in CD8 + T- cells, suggesting the observed T-cell expansion with DARZALEX lM (daratumumab) treatment was antigen-driven.
  • DARZALEX IM (daratumumab) caused a reduction in immune suppressive MDSC and regulator ⁇ - T- and B-cells. These reductions were concomitant with an expansion of CD4 + T-helper cells and CD8 + cytotoxic T-cells. T-cell clonality and functional anti-viral responses as measured by IFN- ⁇ production also increased with DARZALEX lM (daratumumab) treatment. These observations indicate that T-cells continued to function properly, despite low CD38 expression, and suggest that increased T-cell response may be due to depletion of regulator)' cells. Further, these changes in T- cell expansion, activity, and clonality were more pronounced in patients who responded to DARZALEX iM (daratumumab) compared with those who did not. Relapse from
  • DARZALEXTM (daratumumab) therapy was associated with reversal of many of these changes. This suggests an additional, previously-uncharacterized mechanism of action of DARZALEX lM (daratumumab) through immunomodulation that may contribute to clinical responses and its efficacy.
  • Antibodies that promote antitumor immune responses rather than targeting the cancer directly, have demonstrated efficacy in a range of settings.
  • Antibodies inhibiting CTLA-4 and PD-1 promote T-cell expansion and enhance T-celi activation, resulting in prolonged survival and delayed disease recurrence in patients with advanced solid tumors and hematologic malignancies such as Hodgkin lymphoma.
  • these immunomodulatorv' antibodies may not only induce clinical responses, but also prevent disease recurrence.
  • Peripheral blood samples were collected in standard serum separator tubes (2.5 mL to 5 mL) and serum aliquots were shipped frozen SomaLogic, Inc (Boulder, CO) for miiitianalyte serum protein profiling.
  • the serum protein profiling was performed at SomaLogic using a pre -validated SOMAscan assay that measures 1129 protein anaiytes by use of SOMAmer affinity based molecules.
  • SOMAmer reagents are single stranded DNA -based protein affinity reagents. The assay uses small amounts of input sample (150 uL plasma) and converts the protein signal to a SOMAmer signal that is quantified by custom DNA microarray.
  • Each SOMAmer contains 4 functional moieties:
  • the unique protein recognition sequence uses DNA and incorporates chemically modified nucleotides that mimic amino acid side chains, expanding the diversity of standard aptamers and enhancing the specificity and affinity of protein-nucleic acid interactions (Gold et a!., PLoS One 5:el5004, 2010) .
  • the aptamers are selected for by SELEX.
  • SOMAmer reagents are selected using proteins in their native conformations. As such SOMAmer reagents require an intact, tertiary protein structures for binding.
  • Master mixes of SOMAmer reagents are grouped for sample type and dilution.
  • the reagents are pre-bound to streptavidin beads prior to sample incubation. Proteins in the samples are bound to the cognate SOMAmers during equilibrium, washed, incubated with NHS-biotin, washed and then the beads are exposed to UV light to cleave the photocleavable linker.
  • the elution contains the SOMAmer reagents bound to their biotin labeled proteins.
  • a streptavidin capture and subsequent washes removes the unbound SOMAmer reagents.
  • the SOMAmer molecules are released from their cognate proteins through denaturing conditions.
  • the final eluate is hybridized to custom Agilent DNA microarrays and the fluorophore from the SOMAmer molecules it quantified by relative fluorescent units (RFU).
  • the RFU is proportional to the amount of protein in the sample.
  • Samples from the MMY2002 study were tested in two primary batches.
  • a first batch of 180 samples contained paired Cycle 1 Day 1 (ClDl, baseline) and C3D1 (Cycle 3 Dayl) serum samples from 90 subjects.
  • the 180 samples were analyzed together on 3 separate SomaScan plates.
  • the second batch of samples includes 50 ClDl samples, including 35 repeated samples from batch 1.
  • responders are defined as subjects with an overall best response (per IRC, for MMY2002) of sCR, VGPR, and PR, stable disease (SD) subjects as a subject with minimal response (MR) or SD, and non-re sponders are defined as subjects with an o verall best response (per IRC, for MMY2002) of progressi ve disease (PD).
  • IRC overall best response
  • VGPR VGPR
  • PR stable disease
  • MR minimal response
  • non-re sponders are defined as subjects with an o verall best response (per IRC, for MMY2002) of progressi ve disease (PD).
  • SomaLogic's standard inter-plate calibration workflow by defining plate-wide calibration scaling factors for each SOM Amer by calculating the ratio of a Master-mix specific global reference value to the median of 7 in-plate control calibrator measurements.
  • the plate - specific scaling factor for each SOMAmer reagent was applied to each sample on the plate equivalently.
  • Baseline protein level MMY2002 data was used to build a response prediction classifier.
  • SVM Support Vector Machines
  • RF Random Forests
  • NB Naive Bayes
  • j48 decision trees For each learner, the training procedure began with creating 10 balanced folds of the dataset (outer loop). One of these folds was held out as a test cohort while the remaining 9 were passed to an inner loop as the training cohort. Within the inner loop, the training cohort was once again split into 10 balanced folds, creating inner-training and inner-test sets. Learners were trained on each of these inner-training sets and this process was repeated 30 times for each cohort within the outer loop.
  • FIG. 18 shows protein expression profile of PD-L1 in responders, non-responders and in patients with stable disease at cycle 1 and cycle 3.
  • PD-L1 engagement with its receptor PD-1 suppresses anti-tumor responses and drives T cell anergy and exhaustion. While not wishing to be bound by any particular theory, downregulation of PD-L1 upon CD38 treatment may also result in improved potentiation of anti-tumor immune responses in solid tumors.

Abstract

The present invention relates to methods of immunomodulation and treating patients having solid tumors with antibodies that specifically bind CD38.

Description

IMMUNE MODULATION AND TREATMENT OF SOLID TUMORS WITH ANTIBODIES THAT SPECIFICALLY BIND CD38
FIELD OF THE INVENTION
The present invention relates to methods of immune modulation and treatment of solid tumors with antibodies that specifically bind CD38.
BACKGROUND OF THE IN VENTION
The immune system is tightly controlled by a network of costimulatory and co- inhibitory ligands and receptors. These molecules provide secondaiy signals for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection and tumors, while limiting immunity to self (Wang et al, (Epub Mar. 7, 201 \) JExp Med 208(3):577-92; Lepenies et al , (2008) Endocr Metah Immune Disord Drug Targets 8:279-288).
Immune checkpoint therapy to treat solid tumors, targeting co -inhibitor}' pathways in T cells to promote antitumor immune responses, has led to advances in clinical care of cancer patients with approval of anti-CTLA-4 and anti-PD-1 antibodies
YERVOY J (ipilimurnab), KEYTRUDA* (pembrolizumab) and OPDIVO*1 (nivolumab). While anti-PD-l/PD-Ll antibodies are demonstrating encouraging clinical responses in patients with multiple solid tumors, the response rates are still fairly low, about 15% - 20% in pretreated patients (Swaika et al, (2015) Mol Immunol doi:
10.1016/j .molimm.2015.02.009).
While natural killer cells (NK), dendritic cells (DC) and effector T cells are capable of driving potent anti -tumor responses, tumor cells often induce an
immunosuppressive microenvironment, favoring the development of immunosuppressive populations of immune cells, such as myeloid-derived suppressor cells (MDSC), regulatory T-cells (Treg) or regulatory B-ceils (Breg), which contribute to tumor immune tolerance and the failure of immunotherapy regimens in cancer patients and experimental tumor models.
Thus, there remains a need to develop new cancer immunotherapies that induce adaptive immune response against tumors or target the immunosuppressive immune cells. SUMMARY OF THE INVENTION
The invention provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38.
The invention also provides a method for treating a patient having a regulator ' T- cell (Treg) mediated disease comprising administering to the patient in need thereof a therapeutically effecti ve amount of an antibody that specifically binds CDS 8.
The invention also provides a method for treating a patient having a myeloid- derived suppressor cell (MDSC) mediated disease comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8.
The invention also provides a method for treating a patient having a regulatory B~ cell (Breg) mediated disease comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8.
The invention also provides a method of suppressing activity of a regulatory T-ceil (Treg), comprising contacting the Treg with an antibody that specifically binds CDS 8.
The invention also provides method of suppressing activity of a myeloid-derived suppressor cell (MDSC), comprising contacting the MDSC with an antibody that
specifically binds CDS 8.
The invention also provides a method of suppressing activity of a regulatory B- cell (Breg), comprising contacting the Breg with an antibody that specifically binds CD38.
The invention also provides a method of enhancing an immune response in a patient, comprising administering to the patient an antibody that specifically binds CDS 8.
The invention also provides a method of treating a patient having a solid tumor comprising reducing the number of Tregs cells in the patient by administering to the patient an antibody that specifically binds CDS 8.
The invention also provides a method of treating a patient having a solid tumor, comprising reducing the number of myeloid-deri ved suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds CD38.
The invention also provides a method of of suppressing activity of an immune suppressor cell, comprising contacting the immune suppressing cell with an antibody that specifically binds CDS 8.
The invention also provides a method of treating a patient having a viral infection, comprising administering to the patient in need thereof an antibody that specifically binds CDS 8. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows that the median number of lymphocytes was increased in patients responding to DARZALEX™ (daratumumab) treatment at 8 mg kg (upper line) or 16 ltig/kg (Sower line) doses over time, and that the lymphocyte numbers returned to baseline after end of treatment. Study: SIRIUS. X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (C lDl : cycle I, day 1: C1D4; cycle 1, day 4, etc). SCR: baseline; EOT: end of treatment; WK: week; POST-WK: post-treatment at indicated weeks; post-PD FU: follow-up after progression. The highlighted areas in gray shades indicate the 25-27% Interquartile Range (IQR) for the data points for each visit for responders.
Figure 2 shows the percent (%) change of absolute counts of CDS " T cells to baseline in peripheral blood in patients treated with DARZALEX™ (daratumumab) for each individual patient (light gray lines). Study: SIRIUS (MMY2002). The X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (ClDl : cycle 1, day 1; C1D4; cycle 1, day 4, etc). WK: week; POST-WK: post-treatment at indicated weeks;
POST-PD FU: follow-up after progression. The black line shows the median % change for all patients.
Figure 3 shows the percent (%) change of absolute counts of CD4+ T cells to baseline in peripheral blood in patients treated with DARZALEX [M (daratumumab) for each individual patient (light gray lines). Study: SIRIUS. The X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (Cl Dl : cycle 1, day 1 ; CID4; cycle 1 , day 4, etc). WK: week; POST-TMT: post-treatment. The black line shows the median % change for ail patients.
Figure 4 shows the percent (%) change of absolute counts of CD8+ T cells to baseline in peripheral blood in patients treated with DARZALEX™ (daratumumab) for each individual patient (light gray lines). Study: SIRIUS. The X-axis indicates time expressed as ireatmeni cycle and days of dosing within each treatment cycle (ClDl : cycle 1, day 1; C 1D4; cycle 1, day 4, etc). WK: week; Pre-PD FU: follow-up before progression; Post-PD FU: follow-up after progression. The black line shows the median % change for all patients.
Figure 5 shows that the number of CD45XD3+ cells (measured as percentage of
lymphocytes) in bone marrow aspirates was increased during DARZALEX lM (daratumumab) treatment ove time at doses 8 mg/kg or 16 mg/kg. The graph includes both responders and non-responders as indicated. Study: SIRIUS. The X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (C2D22: cycle 2, day 22; etc). SCR: baseline; Post-PD FUI : follow-up after progression. The highlighted areas in gray shade indicate the 25-27% Interquartile Range (IQR) for the data points for each visit for the non-responders dosed at 8 mg/kg, the responders dosed at 16 mg/kg, or the non -responders dosed at 16 mg/kg, respectively. NR: no-responder; R: responder.
Figure 6 shows that the number of CD45 rCD3+ CD81 cells (measured as percentage of lymphocytes) in bone marrow aspirates was increased during DARZALEX lM (daratumumab) treatment over time at doses 8 mg/kg or 16 mg/kg. The graph includes both responders and non-responders as indicated. Study: SIRIUS. The X-axis indicates time expressed as treatment cycle and days of dosing within each treatment cycle (C2D22: cycle 2, day 22; etc). SCR: baseline; Post-PD FU 1 : follow-up after progression. The highlighted areas in gray shade indicate the 25-27% Interquartile Range (IQR) for the data points for each visit for the non-responders dosed at 8 mg/kg, the responders dosed at 16 mg/kg, or the non-responders dosed at 16 mg/kg, respectively. NR: no-responder; R: responder.
Figure 7 A shows that the ratio of CD87 Treg and CD87CD4+ cells in peripheral blood expressed as median values of all treated patients increased over time during DARZALEX1*1 (daratumumab) treatment. Time points: C 1D1 : cycle 1 day; C3D 1 : cycle 3 day 1 ; C4D 1 : cycle 4 day 1 . Study: SIRIUS. SRC: baseline.
Figure 7B shows that the ratio of CD87Treg cells in bone marrow aspirates expressed as median values of all treated patients increased over time during DARZALEXlM
(daratumumab) treatment. Time points: C 1D 1 : cycle 1 day: C3D 1 : cycle 3 day 1 ; C4D 1 : cycle 4 day 1. Study: SIRIUS.
Figure 8A shows that responders had increased CD8+ T-cell cionality when compared to non-responders, as measured using % change in abundance (CIA) of particular clonal cells. Study: GEN501 17 patient subset.
Figure 8B shows the fold change in CD8+ T-cell cionality in individual patients pre- vs. post DARZALEX lM (daratumumab) treatment. Responders are indicated with the star. Cionality was measured as fold change in abundance (CIA) of particular clonal cells. Study: GEN501 17 patient subset.
Figure 8C shows that responders (Group A) had greater total expansion in the TCR repertoire, measured using CIA (change in abundance) when compared to non-responders (Group B). P 0.037. Study: GEN501 17 patient subset.
Figure 8D shows the sum of absolute change in abundance (CIA) in responders and non- responders for each expanded T cell clone. P=0.035 between responders (Group A) and non- responders (Group B). Study: GEN501 17 patient subset.
Figure 8E shows the maximum CIA of a single T-cell clone in responders (Group A) and non-responders (Group B). Study : GEN501 17 patient subset. Figure 8F shows that responders (Group A) had greater maximum expansion of a single clone, measured using maximum % CIA, when compared to non -responders (Group B). P 0.0477. Study: GEN501 17 patient subset.
Figure 9A shows the percentage (%) of CD8+ naive ceils in peripheral blood in non- responders (NR, black squares) and in patients having at least minimal response (MR, white squares) to DARZALEXIM (daratumumab) at baseline, or at 2 weeks, 4 weeks or 8 weeks of treatment, or after relapse. Study: GEN501 17 patient subset. **p=1.82 x 10"4.
Figure 9B shows the percentage of CD8+ central memory cells (Tern) in peripheral blood in non-responders (NR, black squares) and in patients having at least minimal response (MR, white squares) to DARZALEX lM (daratumumab) at baseline, or at 2 weeks, 4 weeks or 8 weeks of treatment, or after relapse. Study: GENS 01 17 patient subset. *p= .88xl0~ .
Figure 9C shows the percentage increase of HLA Class I restricted CD¾"r T cells in peripheral blood at baseline, or at week 1 , 4 or 8 of treatment, or after relapse. Study: GEN501 17 patient subset.
Figure 9D shows that CDS 8 is expressed at low levels in CD8+ naive T cells and in CD8 " central memory cells (Tern) in peripheral, blood at bassline or on treatment. Study: GEN501 17 patient subset. MFT: Mean fluorescent intensity.
Figure 10A shows a histogram of FACS analyses showing frequency of Tregs (CD3T CD3+CD4+CD25+CD127d,tn (top histogram, P4 cell population) and the frequency of CD38 Tregs within the 'Treg population (bottom histogram, P5 cell population) in multiple myeloma patients at baseline. Study: GEN501 17 patient subset- Figure 10B shows a histogram of FACS analyses showing frequency of Tregs (CD3" CD3 "CD44"CD25"tCD127dim (top histogram, P4 cell population) and the frequency of CD38 " Tregs within the Treg population (bottom histogram, P5 cell population) in multiple myeloma patients after DARZALEXlM (daratumumab) treatment. DARZALE lM (daratumumab) treatment depleted CD38" Tregs. Study: GEN501 17 patient subset.
Figure IOC shows that frequency of the CD38M h CD3†CD4 iCD25+CD127dils Tregs in patients treated with DARZALEX™ (daratumumab) at baseline, or at 1 week, 4 week, 8weeks, after relapse or at end of treatment at 6 months (EOT), CD38tasa Treg frequency was reduced with DARZALEX lM (daratumumab) treatment and returned to baseline at EOT. Y- axis: % of CD38i,ighCD3 iCD4†CD25+CD127dlls Tregs from CD3 " T-cells. Study: GEN501 17 patient subset.
Figure 10D shows the CD87Treg cell ratio in responders and non-responders at baseline, at 1 week, 4 weeks and 8 weeks of treatment. The CD8+/Treg cell ratio was significantly higher in responders vs. non-responders (p=0.00955) at Week 8 of treatment. Study: GEN501 17 patient subset.
Figure 10E shows that effector cell proliferation is inhibited more efficiently in the presence of CD38 r Tregs when compared to the CD38~ Tregs or negative controls. Error bars represent standard error. Asterisks denote significant changes. Samples were obtained from multiple healthy donors. Cell proliferation was assessed through the dilution of carboxyfluorescein succinimidyl ester (CFSE).
Figure 11 shows that Myeloid-derived suppressor cells (MDSC) are present in multiple myeloma patients (top graph, boxed cells) and that about half of the cells expressed CD38 (middle graph, boxed cells). The CD38high MDSC population was depleted in patients treated with one infusion of DARZALEXiM (daratumumab) (bottom graph, boxed cells). Study: GEN501 17 patient subset.
Figure 12 shows that the number of CD38high MDSCs (CD1 1 b+HLADR"CD 14"
CD33+CD15+) was reduced in patients after 1 week, 4 week or 8 week treatment with DARZALEXJ M (daratumumab) when compared to the baseline, and returned to close to baseline after end of treatment (EOT). Relapsed patients still demonstrated reduced CD38high MDSCs. Black squares: non-responders; white squares: patients with at least Minimal Response to DARZALEX1J l (daratumumab) treatment. The vertical lines indicate the median values in each group. Patients 2, 4, 15, 16 and 17 demonstrated high initial CD38high MDSCs population. Study: GEN501 17 patient subset.
Figure 13 shows that the patients with highest CD38high MDSCs (patients 2, 4, 15, 16 and 17) had the highest Progression-Free Survival (PFS). These patients had either partial Response (PR) or Minimal Response (MR) to DARZALEX [M (daratumumab) treatment. SD : Stable Disease; PD : Progressive Disease. X-axis shows the PFS for each individual numbered patient.
Figure 14 shows that MDSC are sensitive to DARZALEXlM (daratumumab)-induced ADCC. Daudi ceils were used as positive control for target ceils in the assays. % cell lysis was measured.
Figure ISA shows that CD38+ Bregs were depleted in patients treated with DARZALEX1M (daratumumab) at Week 1, Week 4 and Week 8 of treatment.
Figure 15B shows that CD38+ Bregs secrete lL-10 upon stimulation.
Figure 16A shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN-y production in PBMCs from DARZALEXlM (daratumumab) treated patient with VGPR at baseline and at indicated times during treatment. OD: optical density. White bar: negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only. Asterix indicates a statistically significant change. Pre 4, 8, 10 = Week 4, 8 or 10 of treatment.
Figure 16B shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN-γ production in PBMCs from DA ZALEXlM (daratumumab) treated patient with CR at baseline and at indicated times during treatment. OD: optical density. White bar: negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only, Asterix indicates a statistically significant change. Pre 4, 8, 10 = Week 4, 8 or 10 of treatment.
Figure 16C shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN-γ production in PBMCs from DA ZALEXlM (daratumumab) treated patient with PD at baseline and at indicated times during treatment. OD: optical density. White bar: negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only, Ns: not significant. Pre 4, 8 =:: Week 4 or 8 of treatment.
Figure 16D shows the anti-viral response measured through CMV, EBV and influenza virus specific (CEF) IFN-γ production in PBMCs from DA ZALEXlM (daratumumab) treated patient with MR at baseline and at indicated times during treatment. OD: optical density. White bar: negative control; black bar: CEF added; dashed bar: allogeneic PBMCs only. Ns: not significant. Pre 4, 8 =:: Week 4 or 8 of treatment.
Figure 16E shows the percentage (%) of proliferating virus-reactive T ceils in PBMCs from
DARZALEX™ (daratumumab) treated patient with VGPR at baseline and at indicated times during treatment. White bar: negative control; black bar: CEF added. Asterix indicates a statistically significant change. Pre 4, 8, 10 = Week 4, 8 or 10 of treatment.
Figure 16F shows the percentage (%) of proliferating virus-reactive T cells in PBMCs from
DARZALEX™ (daratumumab) treated patient with CR at baseline and at indicated times during treatment. White bar: negative control; black bar: CEF added. Asterix indicates a statistically significant change. Pre 4, 8, 10 :=: Week 4, 8 or 10 of treatment.
Figure 17A shows a histogram of FACS analyses showing CD38 expression levels on natural killer cells (NK), monocytes, B cells and T cells from a healthy donor.
Figure 17B shows a histogram of FACS analyses showing CD38 expression levels on plasma cells, natural killer ceils (NK), monocytes, B cells and T cells from a multiple myeloma patient.
Figure 17C shows a comparison of the mean fluorescent intensity (MFI) of CD38 in CD38+ Tregs, Bregs, NK, B cells and T cells from relapsed and refractory multiple myeloma patients, CD38 was expressed at lower level in B cells and T cells when compared to the CD38+Tregs, Bregs and NK cells.
Figure 18 shows that PD-L1 protein is downregulated in PBMC samples from responders (R) and upregulated in non-responders (NR.) over time. SD: stable disease. C1D1 : cycle 1 day 1 ; C3D1, cycle 3, day 1. Y axis shows the log2 protein concentration values.
DETAILED DESCRIPTION OF THE INVENTION
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.
Tims, for example, reference to "a cell" includes a combination of two or more cells, and the like .
"CD38" refers to the human CD38 protein (synonyms: ADP-ribosyl cyclase 1, cADPr hydrolase 1 , cyclic ADP-ribose hydrolase 1). Human CD38 has an amino acid sequence shown in GenBank accession number NP 001766 and in SEQ ID NO: 1. It is well known that CD38 is a single pass type II membrane protein with amino acid residues 1-21 representing the cytosolic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain of CD38.
SEQ ID NO: 1
IVIANCEFSPVSGDKPCCRL-SRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGT T RFPEWLARCVKYTCIHPEMRHVDCQSVWDAFKGAFISKHPCNrreEDYQPLM
KLGTQTVPCNKILLWSRiKDLAIiQFTQ rQRDMFTLEDTLLGYLADDLTVVCGEFN TSKINYQSCPDWR J3CSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDK NSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTTKELESnSKRNIQFSC KNIYRPDKFLQCVKNPEDSSCTSEI
"Antibodies" as used herein is meant in a broad sense and includes
immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, hispecific or rnultispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the
immunoglobulin molecule that comprises an antigen binding site of the required
specificity.
Immunoglobulins may be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes TgA l, IgA2, TgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
"Antibody fragments" refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single ann of an antibody; a domain antibody (dAb) fragment (Ward et a!., Nature 341:544- 6, 1989), which consists of a VH domain. VH and VL domains may be engineered and linked together via a synthetic linker to form various types of single chain antibody designs in which the VH/VL domains pair intramolecularly, or intennolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Intl. Pat. Publ. Nos.
WO 1998/44001, WO1988/01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using well known techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.
"Isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding CD38 is substantially free of antibodies that specifically bind antigens other than human CD38). An isolated antibody that specifically binds CD38, however, may have cross-reactivity to other antigens, such as orthologues of human CD38, such as Macaco, fascicidaris (cynomolgus) CD38. In case of a bispecific antibody, the bispecific antibody specifically binds two antigens of interest, and is substantially free of antibodies that specifically bind antigens other that the two antigens of interest. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. '"Isolated antibody" encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure. "Specific binding" or "specifically binds" or "binds" refers to an antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the antibody binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (KD) of about IxlO"8 M or less, for example about IxlO"9 M or less, about 1x10"10 M or less, about lxl 0" S 1 M or less, or about IxlO"12 M or less, typically with the KD that is at least one hundred fold less than its KD for binding to a nonspecific antigen (e.g., BSA, casein). The dissociation constant may be measured using standard procedures. Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Calhthrix jacchus (common marmoset, marmoset). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.
An antibody variable region consists of a "framework" region interrupted by three "antigen binding sites". The antigen binding sites are defined using various terms:
Complementarity Determining Regions (CDRs), three in the VH (HCDR1 , HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat (1970) J Exp Med 132:211-50: Kabat et a! Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); "Hypervanable regions", "HVR", or "HV", three in the VH (HI , H2, H3) and three in the VL (L I, L2, L3) refer to the regions of antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk (1987) A:io/ /¾o/ 196:901-17). Other terms include "IMGT-CDRs" (Lefranc et al, (2003) Dev Comparat Immunol 27:55-77) and "Specificity Determining Residue Usage" (SDRU) (Almagro (2004) Mo I Recognit 17: 132-43). The International ImMunoGeneTics (IMGT) database (http:/7www imgt org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al, (2003) Dev Comparat Immunol 27:55-77.
"Chothia residues" as used herein are the antibody VL and VH residues numbered according to Al-Lazikani (AI-Lazikani et al, ( 1997) JMol Biol 273:927-48).
"Framework" or "framework sequences" are the remaining sequences of a variable region other than those defined to be antigen binding sites. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined. "Humanized antibody" refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.
''Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin.
A human antibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci. A "human antibody" may contain amino acid differences when compared to the human gennline immunoglobulin or rearranged immunoglobulin genes due to for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding site, or both. Typically, "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in ammo acid sequence to an amino acid sequence encoded by human gennline immunoglobulin or rearranged immunoglobulin genes. In some cases, "human antibody" may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et at, (2000) JMol Biol 296:57-86, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et ai, (2010) JMol Biol 397:385-96, and Intl. Pat. Publ. No.
WO2009/085462.
Human antibodies derived from, human immunoglobulin sequences may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody gennline repertoire in vivo.
Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of human antibody. "Recombinant antibody" includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal, for example a mouse or a rat that is transgenic or transchromosomai for human
immunoglobulin genes or a hybridoma prepared therefrom (described further below), antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.
"Monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes. "Monoclonal antibody" therefore refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of Oterminai lysine from the antibody heavy chain. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.
"Epitope" means a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may- have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and/or noncontiguous amino acids that form a conformational spatial unit. For a noncontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3- dimensional space through the folding of the protein molecule.
"V ariant" refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.
"In combination with" means that two or more therapeutics are administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. "Treat" or "treatment" refers to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological change or disease, such as the development or spread of tumor or tumor ceils, or to provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, lack of metastasis, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
'Treatment" may also mean prolonging survival as compared to expected survival if a subject was not receiving treatment. Those in need of treatment include those subjects already with the undesired physiological change or disease as well as those subjects prone to have the physiological change or disease.
"Therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. Exemplar}' indicators of an effective therapeutic or combination of therapeutics include, for example, improved well-being of the patient, reduction in a tumor burden, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer ceils to other locations in the body.
"Inhibits growth" (e.g. referring to tumor cells) refers to a measurable decrease or delay in the tumor cell growth or tumor tissue in. vitro or in vivo when contacted with a therapeutic or a combination of therapeutics or drugs, when compared to the decrease or delay in the growth of the same tumor ceils or tumor tissue in the absence of the therapeutic or the combination of therapeutic drags. Inhibition of growth of a tumor cell or tumor tissue in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
"Regulatory T cells" or "Tregs" or "Treg" refers to T lymphocytes that regulates the activity of other T cell(s) and/or other immune cells, usually by suppressing their activity. The Tregs may be CD3+CD4+CD25+CD127dimT cells, it is appreciated that the Tregs may not be fully restricted to this phenotype, and may express Foxp3.
"Effector T cells" or "Teffs" or "Teff ' refers to T lymphocytes that cany out a function of an immune response, such as killing tumor cells and/or activating an antitumor immune-response which can result in clearance of the tumor cells from the body. The Teffs may be CD3+ with CD4T or CD8+. The Teffs may secrete, contain or express markers such as IFN-y, granzyme B and ICOS. It is appreciated that the Teffs may not he fully restricted to these phenotypes.
''Function of Tregs" or "Treg function" refers to a suppressive function of the Tregs that relates to regulation of host immune responses and/or prevention of autoimmunity. Function of Tregs may be suppression of an anti-tumor response elicted by CD8+ T cells, natural killer (NK) cells, M0 cells, B cells, or dendritic cells (DCs), or suppression of proliferation of effector T cells.
"'Inhibit function of Tregs" or "inhibit Treg function" refers to decreasing the level of function of Tregs in vitro or in vivo in an animal or human subject, which may be determined by conventional techniques known in the art. The level of the function of Tregs may be decreased by, for example, at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%. "Inhibit function of Tregs" include reducing the number of Tregs, for example by killing the Tregs via antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC).
"Myeloid-derived suppressor cells" or "MDSCs" or "MDSC" refers to a specialized population of cells that are of the hematopoietic lineage and express the macrophage/monocyte marker CD1 lb and the granulocyte marker Gr~l/Ly~6G.
Phenotype of the MDSCs may be for example CD1 lb+HLA-DR"CD14"CD33+CD 15+. The MDSCs express low or undetectable expression of the mature antigen presenting cell markers MHC Class II and F480. The MDSCs are immature cells of the myeloid lineage and may further differentiate into several cell types, including macrophages, neutrophils, dendritic cells, monocytes or granulocytes. The MDSCs may be found naturally in normal adult bone marrow of human and animals or in sites of normal hematopoiesis, such as the spleen.
"Inhibit function of MDSCs" or "inhibit MDSC function" refers to decreasing the level of function of MDSCs in vitro or in vivo in an animal or human subject, which may be determined by conventional techniques known in the art. The level of the function of MDSC may be decreased by, for example, at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%. "Inhibit function of MDSC" include reducing the number of MDSC, for example by killing the MDSC via antibody effector functions, such as ADCC. The MDSCs may suppress T cell responses such as proliferation, clonal expansion or cytokine production by various mechanisms such as production of reactive oxygen species, peroxynitrites, increased arginase metabolism due to high levels of arginase, and increased nitrous oxide synthase. The MDSCs may response to IFN-γ and several cytokines such as IL-4 and IL-13. IFN-γ may activate MDSCs which induces the activity of nitric-oxide synthase 2 (NOS2). Alternately, Th2 cytokines such as interleukin-4 (IL-4) and IL-13 may activate MDSCs which may lead to the induction of arginase-1 (ARG1) activity. The metabolism of L-arginine by either NOS2 or ARG1 may lead to the inhibition of T-cell proliferation, and the activity of both enzymes together can result in T-cell apoptosis through the production of reactive nitrogen-oxide species.
"Treg related disease" refers to a disease or disorder linked to T regulatory cells (Tregs). Treg related disease may be caused by Treg function, for example, suppression of an anti-tumor response or suppression of effector T cell proliferation. The Treg mediated disease may be cancer. "Treg related disease" and "Treg mediated disease" are used exchangeable herein.
"Enhance respon se of effector T cells" or "enhance T cell responses" refers to enhancement or stimulation of effector T cells in vitro or in vivo in an animal or human subject to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T-celis. Exemplary T-cell responses are proliferation, secretion of γ- interferon from CD 8+ T-cells, antigen responsiveness, or clonal expansion. The manner of measuring this enhancement is known to one of ordinary skill in the art.
"MDSC related disease" refers to a disease or disorder linked to myeloid-derived suppressor cells (MDSCs). MDSC related disease may be caused by a MDSC function, for example, suppression of an anti -tumor response or effector T cell proliferation . The MDSC mediated disease may be cancer. "MDSC related disease" and "MDSC mediated disease" are used exchangeably herein.
"Regulator - B cell" or "Breg" or "Bregs" refers to B lymphocytes that suppress immune responses. The Bregs may be CD 19 D24 D38+ cells, and may suppress immune responses by inhibiting T cell proliferation mediated by IL-10 secreted by the Bregs. It is appreciated that other Breg subsets exists, and are described in for example Ding et a!., (2015) Human Immunology 76: 615-621.
"Breg related disease" refers to a disease or disorder linked to regulator}' B cells. Breg related disease may be caused by for example Breg mediated suppression of an antitumor response or effector T cell proliferation. The Breg mediated disease may be cancer. "Breg related disease" and "Breg mediated disease" are used exchangeably herein.
"Patient" includes any human or nonhuman animal. "Nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians, reptiles, etc. "Patient" and "subject" are used interchangeably herein. The invention provides a method for treating a patient having a solid tumor with an antibody that specifically binds CDS 8 regardless whether the tumor cells express CDS 8 or not. The invention further provides methods for treating a patient having regulatory T cell (Treg), myeloid-derived suppressor cell (MDSC) or regulatory B cell (Breg) mediated disease. The invention further provides methods for modulating Treg, MDSC or Breg activity to treat solid tumors that are CDS 8 positive and/or associated with high levels of these immune suppressive cells.
The invention is based, at least in part, on the discover}- that the anti-CD38 antibody DARZALEX1M (daratumumab) has an immunomodulatory activity in patients, reducing the number of immune suppressive Tregs, MDSCs and Bregs, increasing the number of CDS''' T cells and the ratio of CD8 " to Tregs, promoting CD8+ central memory cell formation and increasing T cell cionality.
DARZALEX1M (daratumumab) and other anti-CD38 antibodies are being evaluated in the clinic for their efficacy to treat heme malignancies and plasma cell disorders, including multiple myeloma, by the a bility of the antibody to eliminate CDS 8- positive cells by antibody effector functions, such as ADCC, CDC, ACDP and apoptosis, but their immunomodulatory activity in promoting adaptive immune responses has not been recognized. Other immune modulator}' antibodies (anti-PDl, anti-CTLA4) function through targeting components of the immune system that suppress anti-tumor responses. For example, anti-PDl antibodies have been demonstrated to increase T-cell proliferation, stimulate antigen-specific memory responses, and partially relieve Treg-mediated suppression of effector T cells in vitro (for example, see U.S. Pat. No. 8,779,105). Two anti-PD-1 antibodies are currently approved for treatment of melanoma, OPDIVO® (nivolumab) and KEYTRUDA© (pembrolizumab) and these antibodies are in clinical development for various solid tumors, such as lung non-small cell carcinoma, prostate, head and neck, gastrointestinal, stomach, prostate, fallopian tube, ovarian, pancreatic, breast and brain cancer, renal, bladder, urethral, oesophageal and colorectal cancer. Anti- CTLA-4 antibody YERVOY© (ipilimumab) has been approved for treatment of melanoma. YERVOY® (ipilimumab) and another anti-CTLA-4 antibody, tremelimumab are also being developed for prostate, non-small cell lung cancer, ovarian, gastrointestinal, stomach, colorectal, renal, oesophageal, and genitourinary cancer.
Without wishing to be bound by any particular theory, based on the
immunomodulatory effects observed with DARZALEX1M (daratumumab) described herein, DARZALEXl (daratumumab) and other anti-CD38 antibodies may be efficacious in treatment of solid tumors. Due to the general acti vation of immune response observed in patients treated with DARZALEXlM (daratumumab), patients having CD38-negative solid tumors may respond to anti-CD38 antibody therapies as well.
The invention provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a regulatory T cell (Treg) mediated disease, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 for a time sufficient to treat the Treg mediated disease.
The invention also provides for a method of treating a patient having a myeloid- derived suppressor cell (MDSC) mediated disease, comprising administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CDS 8 for a time sufficient to treat the MDSC mediated disease.
The invention also provides for a method of treating a patient having a regulatory B cell (Breg) mediated disease, comprising administering to the patient in need tliereof a therapeutically effective amount of an antibody that specifi cally binds CD38 for a time sufficient to treat the Breg mediated disease.
The invention also provides for a method of suppressing activity of a regulatory T cell (Treg), comprising contacting the regulatory T cell with an antibody that specifically binds CD38,
The invention also provides for a method of suppressing activity of a myeloid- derived suppressor cell (MDSC), comprising contacting the MDSC with an antibody that specifically binds CD38.
The invention also provides for a method of suppressing activity of a regulator}' B cell (Breg), comprising contacting the Breg with an antibody that specifically binds CDS 8.
The invention also provides for a method of treating a patient having a solid tumor, comprising reducing the number of regulatory T ceils (Treg) in the patient by administering to the patient an antibody that specifically binds CD38.
The invention also provides for a method of treating a patient having a solid tumor, comprising reducing the number of myeloid -derived suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds CDS 8.
The invention also provides for a method of treating a patient having a solid tumor, comprising reducing the number of regulatory B cells (Breg) in the patient by- administering to the patient an antibody that specifically binds CDS 8. The invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof an antibody that specifically binds CD38 for a time sufficient to enhance the immune response.
In some embodiments, the patient has a viral infection.
The invention also provides for a method of treating a viral infection in a patient, comprising administering to the patient in need therefor an antibody that specifically binds CD38 for a time sufficient to treat the viral infection.
In some embodiments, the immune response is an effector T cell (Teff) response.
In some embodiments, the Teff response is mediated by CD4+ T cells or CD8" T cells.
In some embodiments, the Teff response is mediated by CD4 f T cells.
In some embodiments, the Teff response is mediated by CD8 r T cells.
In some embodiments, the Teff response is an increase in the number of CDS" T cells, increased CD8+ T cell proliferation, increased T cell clonal expansion, increased CD8 " memory cell formation, increased antigen -dependent antibody production, or increased cytokine, chemokine or interleukin production.
Proliferation of T cells may be assessed for example by measuring the rate of DNA synthesis using tritiated thymidine or measuring production of interferon -y (IFN-γ) in vitro, or measuring absolute number or percentage of T cells in a population of cells from patient samples using known methods.
Clonal expansion may be assessed by for example sequencing TCR from a pool of T cells using know methods.
Memory cell formation may be assessed by measuring the ratio of naive T cells (CD45R07CD62L ) to memory T ceils (CD45RO+/CD62L gh) using for example FACS.
Cytokine, chemokine or interleukin production, such as production of interferon -γ (IFN-γ), tumor necrosis factor-alpha (TNF-a), IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL- 12, IL 3, IL-10, IL-18 and IL-23, ΜΊΡ-Ια, ΜΙΡ-Ιβ, RANTES, CCL4 may be assessed using standard methods such as EL1SA or ELLISPOT assay.
Antigen-specific antibody production may be assessed from samples derived from patient using standard methods, such as ELISA or radioimmunoassay (RTA).
The meaning of "increase" or "increasing" various Teff responses is readily understood. The increase may be increase of at least about 5%, at least about 10%, 25%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400% or more in a test sample or in a subject when compared to control, e.g., for example in a patient treated with an anti-CD38 antibody when compared to the same patient before treatment, or in a patient or group of patients that are responsive to anti-CD38 antibody treatment when compared to a patient or a group of patients that are non-responsive to the same treatment. Typically, the increase is statistically significant.
Similarly, the meaning of "reduce"' or "reducing" or "decreasing" or "decrease" the number of Tregs, MDSCs and/or Bregs is readily understood. The decrease may be at least about 10%, 25%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 1 10%, 120%, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400% or more in a test sample or in a subject when compared to control, e.g., for example in a patient treated with an anti-CD38 antibody when compared to the same patient before treatment, or in a patient or group of patients that are responsive to anti-CD38 antibody treatment when compared to a patient or a group of patients that are non-responsive to the same treatment. Typically, the decrease is statistically significant.
in some embodiments, the antibody that specifically binds CD 38 inhibits function of immune suppressor cells.
Tn some embodiments, the immune suppressor cells are regulatory- T cells (Tregs), myeloid-derived suppressor cells (MDSC) or regulator}' B cells (Bregs).
In some embodiments, the Tregs are CD3+CD4+CD25+CD127d,tn T cells.
In some embodiments, the CD3 rCD4+CD25+CD127aiBI cells express Foxp3.
In some embodiments, the CD3+CD4+CD25÷CD127diin T cells express CD38.
Treg function, such as their ability to suppress Teff cells, may be assessed using known methods, such as assessing the ability of Tregs to suppress Teff proliferation in mixed lymphocyte reaction (MLR).
Treg function may be inhibited by for example reducing the relative number of Tregs when compared to Teffs (e.g. increasing the ratio of CDSVTreg cells) by direct killing of Tregs or a sub-population of Tregs, such as CD38 " Tregs.
In some embodiments, the Treg function is inhibited by killing the Treg cells.
In some embodiments, the Treg killing is mediated by antibody-induced antibody- dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or apoptosis mduced by an antibody specifically binding CD38.
in some embodiment, the Treg killing is mediated by ADCC.
In some embodiments, the CD38+ Tregs are killed.
Tn some embodiments, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of Tregs are killed.
As CD38 is expressed only in a portion of Tregs and MDSCs, it is expected that treatment of patients with solid tumors will not result in systemic depletion of Tregs and MDSCs, therefore likely providing an improved safety profile.
Tn some embodiments, the MDSCs are CD1 lbHLA-DR"CD14"CD33+CD15+ cells.
In some embodiments, the CDl lb+HLA-DR-CD14_CD334'CD15 MDSCs express
CD38.
MDSC function may be inhibited for example by reducing the number of MDSCs by direct killing of the cells.
in some embodiments, the MDSC function is inhibited by killing the CD38
MDSC.
Tn some embodiments, the MDSC killing is mediated by antibody-induced antibody-dependent cell cytotoxicity (ADCC), antibody -dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or apoptosis induced by the antibody that specifically binds CD38.
In some embodiments, the MDSC killing is mediated by ADCC.
In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of the MDSCs are killed.
In some embodiments, the Bregs are CD19†CD24+CD38+ cells.
The Breg function may be inhibited for example by reducing the number of Bregs by direct killing of the Bregs.
In some embodiments, the Breg function is inhibited by killing the CD38" Bregs. In some embodiments, the Breg killing is mediated by antibody-induced antibody- dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement -dependent cytotoxicity (CDC) or apoptosis induced by the antibody that specifically binds CD38,
Tn some embodiments, the Breg killing is mediated by ADCC.
In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of the Bregs are killed.
Tregs play a critical role in the maintenance of peripheral self-tolerance. Naturally occurring CD4TCD25i" Tregs are produced in the thymus and express Foxp3, a transcriptional factor required for establishment and maintenance of Treg lineage identity and suppressor function. Tregs can accumulate at a disease site (e.g. within tumor), where they suppress the effector function of tumor antigen specific T cells, resulting in insufficient anti-tumor responses. Increased densities of tumor-infiltrating Foxp3+ Tregs have been associated with poor prognosis in various solid tumors, including pancreatic, ovarian, and hepatocellular carcinoma. Depletion of Tregs results in enhanced antitumor immunity and tumor rejection in murine models but may also result in the development of autoimmune diseases.
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of early myeloid progenitors, immature granulocytes, macrophages, and dendritic cells at different stages of differentiation. They accumulate in large numbers in cancer patients and they have potent immunosuppressive functions, suppressing both the cytotoxic activities of natural killer cells (NK) and natural killer T cells (NKT), and the adaptive immune response mediated by CD8+ T cells. While the mechanism of NK cell inhibition is currently not well-understood, multiple pathways are responsible for MDSC-mediated T cell suppression including production of arginase 1/ARGl and upregulation of nitri c oxide synthase 2 (NOS2). ARGI and NOS2 metabolize L-arginine and either together or separately blocks the translation of the T cell CD3zeta chain, inhibits T cell proliferation, and promotes T cell apoptosis. Additionally, MDSCs secrete immunosuppressive cytokines and induce regulatory T cell development.
MDSC are induced by pro-inflammatory cytokines and are found in increased numbers in infectious and inflammatory pathological conditions. They accumulate in the blood, bone marrow, and secondary lymphoid organs of tumor-bearing mice and their presence in the tumor microenvironment as been suggested to have a causative role in promoting tumor-associated immune suppression.
MDSC have been described in patients with colon carcinoma, melanoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, non-small cell lung carcinoma, renal cell carcmoma, pancreatic adenocarcinoma and breast carcinoma (Mandruzzato ei al, (2009) J Immunol 182: 6562-6568; Lm et al, (2009) J Cancer Res dm Oncol 136: 35-45; Ko ei al . (2009) Clin Cancer Res 15: 2148-2157; Morse ei al, (2009) Expert Opin Biol Ther 9: 331-339; Diaz-Montero ei al, (2009) Cancer Immunol Immunother 58: 49-59; Corzo et al, (2009) J Immunol 182: 5693-5701), In cancer patients, Diaz et al (Diaz-Montero ei at, (2009) Cancer Immunol Immunother 58: 49-59) propose that accumulation of MDSC correlates with more advanced disease and poor prognosis.
Tumor-infiltrating Bregs have been identified in solid tumors, and the Bregs may promote tumor growth and metastasis by various mechanisms such as suppressing the antitumor activity of CD8 " T ceils and NK cells, as described in for example Ding et al, (2015) Human Immunology 76:615-62.
"Antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or '"ADCC" is a mechanism for inducing cell death that depends upon the interaction of antibody -coated target cells with effector ceils possessing lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells. For example, NK cells express FcyRTIIa, whereas monocytes express FcyRI, FcyRTI and FcvRITIa. Death of the antibody-coated target cell, such as CD38-expressing cells, occurs as a result of effector cell activity through the secretion of membrane pore-forming proteins and proteases. To assess ADCC activity of an antibody that specifically binds CD38, the antibody may be added to CD38 -expressing cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell . Cytolysis is generally detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed ceils. Exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Exemplary target cells include Tregs or MDSCs expressing CD38. In an exemplary assay, target cells are labeled with 20 μθ of 3lCr for 2 hours and washed extensively. Cell concentration of the target cells may be adjusted to 1 χ 106 cells/ml, and anti-CD38 antibodies at various concentrations are added. Assays are started by adding target cells at an effectontarget cell ratio of 40: 1. After incubation for 3 hr at 37°C assays are stopped by centrifugation and 51Cr release from lysed cells are measured in a scintillation counter. Percentage of cellular cytotoxicity may be calculated as % maximal lysis which may be induced by adding 3% perchloric acid to target cells.
"Antibody-dependent cellular phagocytosis" ("ADCP") refers to a mechanism of elimination of antibody -coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells. ADCP may be evaluated by using Tregs or MDSCs expressing CD38 as target cells engineered to express GFP or other labeled molecule. Effctontarget cell ratio may be for example 4: 1. Effector cells may be incubated with target cells for 4 hours with or without anti-CD38 antibody. After incubation, cells may be detached using accutase. Macrophages may be identified with anti-CD 1 lb and anti- CD14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescent in the CD1 1 D14÷ macrophages using standard methods.
"Complement -dependent cytotoxicity", or "CDC1, refers to a mechanism for inducing cell death in which an Fc effector domain of a target-bound antibody binds and activates complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.
The ability of monoclonal antibodies to induce ADCC may be enhanced by engineering their oligosaccharide component. Human IgGl or IgG3 are N-glycosylated at Asn297 with the majority of the glycans in the well-known biantennary GO, GOF, Gl, GIF, G2 or G2F forms. Antibodies produced by non-engineered CHO cells typically have a glycan fucose content of about at least 85%. The removal of the core fucose from the biantennary complex-type oligosaccharides attached to the Fc regions enhances the ADCC of antibodies via improved FcyRllla binding without altering antigen binding or CDC activity. Such niAbs may be achieved using different methods reported to lead to the successful expression of relatively high defucosylated antibodies bearing the biantennary complex-type of Fc oligosaccharides such as control of culture osmolality (Konno et ai, (2012) Cytotechnology 64:249-65), application of a variant CHO line Led 3 as the host cell line (Shields et al, (2002) J Biol Chem 277:26733-26740), application of a variant CHO line EB66 as the host cell line (Olivier et al, (2010) MAbs 2(4), Epub ahead of print; PMID:20562582), application of a rat hybridoma cell line YB2/0 as the host cell line (Shinkawa et al., (2003) J Biol Chem 278:3466-3473), introduction of small interfering RNA specifically against the a 1 ,6-fucosyltrasferase ( FUT8) gene (Mori et ah, (2004) Biotechnol Bioeng 88:901 -908), or coexpression of P-l,4-N-acetylglucosaminyltransferase III and Golgi a-mannosidase II or a potent alpha-mannosidase I inhibitor, kifunensine
(Ferrara et al., (2006) J Biol Chem 281:5032-5036; Ferrara et al, (2006) Biotechnol Bioeng 93:851-861; Xhou et al, (2008) Biotechnol Bioeng 99:652-65). ADCC elicited by anti-CD38 antibodies used in the methods of the invention, and in some embodiments of each and every one of the numbered embodiments listed below, may also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are for example substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EU index) as described in U.S. Pat. No.
6,737,056.
In some embodiments, the antibody that specifically binds CD38 comprises a substitution in the antibody Fc.
In some embodiments, the antibody that specifically binds CD38 comprises a substitution in the antibody Fc at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EU index).
In some embodiments, the antibody that specifically binds CD38 has a biantennary glycan structure with fucose content of about between 0% to about 15%, for example 15%, 14%, 13%, 12%, 11 % 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.
In some embodiments, the antibody that specifically binds CD38 has a biantennary glycan structure with fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 1 1% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, l% or 0%
Substitutions in the Fc and reduced fucose content may enhance the ADCC activity of the antibody that specifically binds CD38.
"Fucose content" means the amount of the fucose monosaccharide within the sugar chain at Asn297. The relative amount of fucose is the percentage of fucose- contaming structures related to ail giycostractures. These may be characterized and quantified by multiple methods, for example: 1) using MALDI-TOF of N-glycosidase F treated sample (e.g. complex, hybrid and oligo- and high-mannose structures) as described in Intl. Pat. Pubi. No. WO2008/077546; 2) by enzymatic release of the Asn297 glycans with subsequent denvatization and detection/ quantitation by HPLC (UPLC) with fluorescence detection and/or HPLC-MS (UPLC-MS); 3) intact protein analysis of the native or reduced mAb, with or without treatment of the Asn297 glycans with Endo S or other enzyme that cleaves between the first and the second GlcNAc monosaccharides. leaving the fucose attached to the first GlcNAc; 4) digestion of the m Ab to constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), and subsequent separation, detection and quantitation by HPLC-MS (UPLC-MS) or 5) separation of the mAb oligosaccharides from the niAb protein by specific enzymatic deglycosylation with PNGase F at Asn 297. The oligosaccharides released may be labeled with a fluorophore, separated and identified by various complementary techniques which allow: fine characterization of the glycan structures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of the experimental masses with the theoretical masses, determination of the degree of sialylation by ion exchange HPLC (GiycoSep C), separation and quantification of the oligosacharride forms according to hydrophilicity criteria by normal-phase HPLC (GiycoSep N), and separation and quantification of the oligosaccharides by high performance capillary electrophoresis-laser induced fluorescence (HPCE-LIF).
''Low fucose" or "low fucose content" as used herein refers to antibodies with fucose content of about 0% - 15%.
"Normal fucose" or 'normal fucose content" as used herein refers to antibodies with fucose content of about over 50%, typically about over 60%, 70%, 80% or over 85%.
In some embodiments, the antibody that specifically binds CD38 may induce killing of Tregs, MDSCs and/or Bregs by apoptosis. Methods for evaluating apoptosis are well known, and include for example annexin IV staining using standard methods. The anti-CD38 antibodies used in the methods of the invention may induce apoptosis in about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of cells.
In some embodiments, the Teffs or the immune suppressor cells reside in bone marrow or in peripheral blood.
In some embodiments, the Teffs or the immune suppressor cells reside in bone marrow.
In some embodiments, the Teffs or the immune suppressor cells reside in peripheral blood.
In some embodiments, the antibody that specifically binds CD38 increases the ratio of CDS 1 T cells to Tregs.
In some embodiments, the antibody that specifically binds CD38 increases the ratio of CD8÷ central memory cells to CD8" naive cells. CD8" central memory cells can be identified as CD45R07CD62Lt sh cells. CD8+ naive cells can be identified as CD45RO- /CD62L4 cells. In some embodiments, the antibody that specifically binds CD38 is a non- agonistic antibody.
A non-agonistic antibody that specifically binds CDS 8 refers to an antibody winch upon binding to CD38 does not induce significant proliferation of a sample of peripheral blood mononuclear cells in vitro when compared to the proliferation induced by an isotype control antibody or medium alone.
In some embodiments, the non-agonistic antibody that specifically binds CD38 induces proliferation of peripheral blood mononuclear cells (PBMCs) in a statistically insignificant manner. PBMC proliferation may be assessed by isolating PBMCs from healthy donors and culturing the cells at 1 x 10s cells/well in flat bottom 96-well plates in the presence or absence of a test antibody in 200 μΐ RPMI After four day incubation at 37° C, 30 μΐ Ή-thymidine (16.7 C'i/mi ) may be added, and culture may be continued overnight. Rhymidine incorporation may be assessed using a Packard Cobra gamma counter (Packard Instruments, Meriden, DT, USA), according to the manufacturer's instructions. Data may be calculated as the mean cpm (±SEM) of PBMCs obtained from several donors. Statistical significance or insignificance between samples cultured in the presence or absence of the test antibody is calculated using standard methods.
An exemplary anti-CD38 antibody that may be used in the methods of the invention is DARZALEX1 M (daratumumab). DARZALEXiM (daratumumab) comprises a heavy chain variable region (VH) and a light chain variable region (VL) amino acid sequences shown in SEQ ID NO: 4 and 5, respectively, a heavy chain complementarity determining region 1 (HCDR1), a HCDR2 and a HCDR3 of SEQ ID NOs: 6, 7 and 8, respectively, and a light chain complementarity determining region 1 (LCDRl), a LCDR2 and a LCDR3 of SEQ ID NOs: 9, 10 and 11 , respectively, and is of IgGl/κ subtype and described in U.S. Pat. No. 7,829,693. DARZALEX!'yi (daratumumab) heavy chain amino acid sequence is shown in SEQ ID NO: 12 and light chain amino acid sequence shown in SEQ ID NO: 13.
In some embodiments, the antibody that specifically binds CD38 competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5.
In some embodiments, the antibody that specifically binds CD38 binds at least to region SKRNIQFSCKNJYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG IQ ID NO: 3) of human CD38 (SEQ ID NO: 1). MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGT
TKJlFPETVLARCVKYTElHPEMRHVDCQSVWDAPKGAFISKFiPCNITEEDYQPLM
KLGTQWPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFN
TSKIN^QSCPDWRKDCS NPVSVFVVKTVSRRFAEAACDVVF MLNGSRSKTFDK NSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSC KN1YRPDKFLQC VKN PEDS SCTSEI
SEQ ID NO: 2
SKRNIQFSCKNIYR
SEQ ID NO: 3
EKV QTLEAWVIHGG
SEQ ID NO: 4
EVQLLESGGGLVQPGGSLRLSCAVSGFTTTSrSFAMS\VVRQAPGKGLEWVSA
ISGSGGGTi^ADSVKGRFTISRDNSKNTLYLQIViNSLRAEDTAWFCAKDK IL FGEPVFDYWGQGTLVTVSS
SEQ ID NO: 5
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA QQKPGQAPRLLr D
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQ GTKVE1K
SEQ ID NO: 6
SFAMS
SEQ ID NO: 7
AISGSGGGTYYADSVKG
SEQ ID NO: 8
DKILWFGEPVFDY
SEQ ID NO: 9
RASQSVSSYLA SEQ ID NO: 10
DASNRAT
SEQ ID NO: 1 1
QQRSNWPPTF
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMS^ RQAPG GLE SAISGSG
GGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVF
DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVtnrPAVLQSSGLYSLSSW PSSSLGTQTYICNAnsiFiKPSNTKVDKRV
EPKSCDKTTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYN^
NK^ LPAPIEKTISKAKGQPREPQVYTLPPS1 EEMTKNQVSLTCLVKGFYPSDL VE
WESNGQPENNYKTTPPVLDSDGSFFLYSKL;rVDKSRW"QQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
SEQ ID NO: 13
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT
GIPARFSGSGSGTDFTLTISSLEPEDFA\'Yl CQQRSN VPPTFGQGTKVEIKRTVAAP SVF1FPPSDEQLKSGTASWCLLNNFYPREAKVQW VDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Antibodies may be evaluated for their competition with a reference antibody such as DARZALEX™ (daratumumab) having the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for binding to CD38 using well known in vitro methods. In an exemplary method, CHO cells recombinantly expressing CD38 may be incubated with an unlabeled reference antibody for 15 min at 4°C, followed by incubation with an excess of
fluorescently labeled test antibody for 45 min at 4°C. After washing in PBS/BSA, fluorescence may be measured by flow cytometry using standard methods. In another exemplar ' method, extracellular portion of human CD38 may be coated on the surface of an ELISA plate. Excess of the unlabeled reference antibody may be added for about 15 minutes and subsequently biotinylated test antibodies may be added. After washes in PBS/Tween, binding of the test biotinylated antibody may be detected using horseradish peroxidase (HRP)-conjugated streptavidin and the signal detected using standard methods. It is readily apparent that in the competition assays, the reference antibody may be labelled and the test antibody unlabeled. The test antibody competes with the reference antibody when the reference antibody inhibits binding of the test antibody, or the test antibody inhibits binding of the reference antibody to CD38 by at least 80%, for example 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The epitope of the test antibody may further be defined for example by peptide mapping or hydrogen/deuterium protection assays using known methods, or by crystal structure determination.
Antibodies binding to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1) may be generated for example by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NOs: 2 and 3 using standard methods and those described herein, and characterizing the obtained antibodies for binding to the peptides using for example ELISA or mutagenesis studies.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof an anti-CD38 antibody that binds to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region
EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). The epitope of the antibody used in the methods of the invention includes some or all of the residues having the sequences shown in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the antibody epitope comprises at least one amino acid in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least one amino acid in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). In some embodiments, the antibody epitope comprises at least two amino acids in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least two amino acids in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). In some embodiments, the antibody epitope comprises at least three ammo acids in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and at least three ammo acids in the region EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
In some embodiments, the antibody that specifically binds CD38 comprises the HCDR1, the HCDR2 and the HCDR3 amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively. In some embodiments, the antibody that specifically binds CDS 8 comprises the LCDR1, the LCDR2 and the LCDR3 ammo acid sequences of SEQ ID NOs: 9, 10 and 1 1, respectively.
In some embodiments, the antibody that specifically binds CD38 comprises the HCDRI , the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 ammo acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10 and 1 1, respectively.
In some embodiments, the antibody that specifically binds CD38 comprises the VH that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 and the VL that is 95%, 96%, 97%, 98% , 99% or 100% identical to SEQ ID NO: 5.
In some embodiments, the antibody that specifically binds CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
In some embodiments, the antibody that specifically binds CD38 comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13.
Other exemplary anti-CD38 antibodies that may be used in any embodiment of the invention are: mAb003 comprising the VH and the VL sequences of SEQ ID NOs: 14 and 15, respectively and described in U.S. Pat. No. 7,829,693. Trie VH and the VL of mAb003 may be expressed as IgGl/κ.
SEQ ID NO: 14
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPF LGIANSAQKFQGRVT1TADKSTSTAY
MDLS SLRSEDTA VYYCARDD IAALGPFD YWGQGTLVTVS SAS
SEQ ID NO: 15
DIQMTQSPSSLSASVGDRVnTCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQS
GVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYNSYPRTFGQGTKVEIK; mAb024 comprising the VH and the VL sequences of SEQ ID NOs: 16 and 17, respectively, described in U.S. Pat. No. 7,829,693. The VH and the VL of mAb024 may be expressed as IgGl/κ. SEQ ID NO: 16
EVQLVQSGAEVKKPGESLiaSC GSGYSFSNYWIGWVRQMPGKGLEWMGIIYPH DSDARYSPSFQGQVTFSADKS1STAY
LQWS SLKASDTAMYYCARHVGWGSRYWYFDLWGRGTLVTV S S
SEQ ID NO: 17
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA\VYQQKPGQAPRLLIYDASNRAT
GIPARFSGSGSGTDFTLTISSLEP
EDFAVYYCQQRSNWPPTFGQGTKVEIK;
MOR-202 (MOR-03087) comprising the VH and the VL sequences of SEQ ID NOs: 18 and 19, respectively, described in US, Pat. No, 8,088,896. The VH and the VL of MOR- 202 may be expressed as IgGl/'κ.
SEQ ID NO: 18
QVQLVESGGGLVQPGGSLI LSCAASGFTFSSYYMNWVRQAPGKGLE VSGISGD PSNTYYADSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS SEQ ID NO: 19
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPS GTPERFSGSNSGNTATLTISGTQAEDEADYYCQ TGGASLVFGGGT LTVLGQ;
Isatuximab: comprising the VH and the VL sequences of SEQ ID NOs: 20 and 21, respectively, described in U.S. Pat. No. 8, 153,765. The VH and the VL of isatuximab may be expressed as IgG l/ .
SEQ ID NO 20:
QVQLVQSGAEVAKPGTSVKLSC ASGYTTTDYWMQWVKQRPGQGLEWIGT
IYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGD YYGSNSLDYWGQGTSVTVSS
SEQ ID NO: 21 :
DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYS ASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGG GTKLEIK
Other exemplary anti-CD38 antibodies that may be used in the methods of the invention include those described in Int. Pat. Publ. No. WO05/103083, Intl. Pat. Publ. No. WO06/125640, Intl. Pat. Publ, No. WO07/042309, Intl. Pat. Publ , No. WO08/047242 or Intl. Pat. Publ. No. WO 14/178820.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, compri sing administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, compri sing administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19 or a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21 or a time sufficient to treat the solid tumor.
In some embodiments, the solid tumor is a melanoma.
In some embodiments, the solid tumor is a lung cancer.
In some embodiments, the solid tumor is a squamous non-small cell lung cancer (NSCLC).
hi some embodiments, the solid tumor is a non-squamous NSCLC.
In some embodiments, the solid tumor is a lung adenocarcinoma.
In some embodiments, the solid tumor is a renal cell carcinoma (RCC) (e.g., a kidney clear cell carcinoma or a kidney papillary cell carcinoma), or a metastatic lesion thereof. In some embodiments, the solid tumor is a mesothelioma.
In some embodiments, the solid tumor is a nasopharyngeal carcinoma (NPC).
In some embodiments, the solid tumor is a colorectal cancer.
In some embodiments, the solid tumor is a prostate cancer or castration-resistant prostate cancer.
In some embodiments, the solid tumor is a stomach cancer.
In some embodiments, the solid tumor is an ovarian cancer.
In some embodiments, the solid tumor is a gastric cancer.
In some embodiments, the solid tumor is a liver cancer.
In some embodiments, the solid tumor is pancreatic cancer.
In some embodiments, the solid tumor is a thyroid cancer.
In some embodiments, the solid tumor is a squamous cell carcinoma of the head and neck.
In some embodiments, the solid tumor is a carcinomas of the esophagus or gastrointestinal tract.
In some embodiments, the solid tumor is a breast cancer.
In some embodiments, the solid tumor is a fallopian tube cancer.
In some embodiments, the solid tumor is a brain cancer.
In some embodiments, the solid tumor is an urethral cancer.
In some embodiments, the solid tumor is a genitourinary cancer.
In some embodiments, the solid tumor is an endometriosis.
In some embodiments, the solid tumor is a cervical cancer.
In some embodiments, the solid tumor is a metastatic lesion of the cancer.
In some embodiments, the solid tumor lacks detectable CD38 expression.
The solid tumor lacks detectable CDS 8 expression when CDS 8 expression in the solid tumor tissue or on cells isolated from the solid tumor is statistically insignificant when compared to a control, e.g. expression detected with anti-CD38 antibody vs expression detected with an isotype control antibody using well known methods.
Anti-CD38 antibodies used in the methods of the invention may also be selected de novo from, e.g., a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions nappik et at, (2000) JMol Biol 296:57- 86; Krebs et at, (2001) J Immunol Meth 254:67-84; Vaughan et al, ( 1996) Nature Biotechnology 14:309-314; Sheets et al, (1998) PITAS (USA) 95:6157-6162;
Hoogenboom and Winter, (1991) J Mol Biol 227 : 381 ; Marks et al. , (1991) JMol Biol 222:581). CD38 binding variable domains may be isolated from e.g., phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al, (2010) JMol Biol 397:385-96, and Intl. Pat. Publ. No. WO09/085462. The antibody libraries may be screened for binding to human CD38 extracellular domain, the obtained positive clones further characterized, Fabs isolated from the clone lysates, and subsequentely cloned as full length antibodies. Such phage display methods for isolating human antibodies are established in the art. See for example: U.S. Pat. No. 5,223,409, U.S. Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,427,908, U.S. Pat. No, 5,580,717, U.S. Pat. No. 5,969,108, U.S. Pat. No. 6,172, 197, U.S. Pat. No. 5,885,793, U.S. Pat. No. 6,521,404, U.S. Pat. No.
6,544,731, U.S. Pat. No. 6,555,313, U.S. Pat. No. 6,582,915, and U.S. Pat. No. 6,593,081.
In some embodiments, the anti-CD38 antibody is of IgGl , IgG2, IgG3 or IgG4 isotype.
The Fc portion of the antibody may mediate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody -dependent cellular phagocytosis (ADCP) or complement dependent cytotoxicity (CDC). Such function may be mediated by binding of an Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity or by binding of an Fc effector domain(s) to components of the complement system. Typically, the effect(s) mediated by the Fc -binding cells or complement components result in inhibition and/or depletion of target cells, for example CD38-expressing cells. Human IgG isotypes IgGl , TgG2, IgG3 and IgG4 exhibit differential capacity for effector functions. ADCC may be mediated by IgGl and IgG3, ADCP may be mediated by IgGl, IgG2, lgG3 and lgG4, and CDC may be mediated by IgGl and IgG3.
Antibodies that are substantially identical to the antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 may be used in the methods of the invention. The term "substantially identical" as used herein means that the two antibody VH or VL amino acid sequences being compared are identical or have "insubstantial differences". Insubstantial differences are substitutions of I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in an antibody heavy chain or light chain that do not adversely affect antibody properties. Percent identity may be determined for example by pairwise alignment using the default settings of the AlignX module of Vector NT! v.9.0.0
(Invitrogen, Carlsbad, CA). The protein sequences of the present invention may be used as a query sequence to perform a search against public or patent databases to, for example, identify related sequences. Exemplary programs used to perform such searches are the XBLAST or BLASTP programs (littp_/Avww_ncbi_nlm/nih_gov), or the GenomeQuest1¾1 (GenomeQuest, Westborough, MA) suite using the default settings. Exemplary substitutions that may be made to the antibodies that specifically bind CDS 8 are for example conservative substitutions with an amino acid having similar charge,
hydrophobic, or stereochemical characteristics. Conservative substitutions may also be made to improve antibody properties, for example stability or affinity, or to improve antibody effector functions. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions may be made for example to the heavy or the light chain of the anti-CD38 antibody. Furthermore, any native residue in the VH or the VL may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis
(MacLennan et al. Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et ciL, Adv Biophys 35: -24, 1998). Desired amino acid substitutions may be determined by those skilled in the art at the time such substitutions are desired. Amino acid substitutions may be done for example by PGR mutagenesis (U.S. Pat. No. 4,683,195). Libraries of variants may be generated using well known methods, for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Tip) and screening the libraries for variants with desired properties. The generated variants may be tested for their binding to CD38, their ability to induce ADCC, ADCP or apoptosis, or modulate CDS 8 enzymatic activity in vitro using methods described herein.
Tn some embodiments, the antibody that specifically binds CD38 may bind human CD38 with a range of affinities (KD). In one embodiment according to the invention, and in some embodiments of each and every one of the numbered embodiments listed below, the antibody that specifically binds CD38 binds to CD38 with high affinity, for example, with a KD equal to or less than about 10"' M, such as but not limited to, 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) x lO"8 M, 10"9 M, 10"10 M, 10"u M, 10"S2 M, 10"lj M, 10"14 M, 10"L M or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art. One exemplary affinity is equal to or less than 1 x10 s M, Another exemplary' affinity is equal to or less than 1x10 " M.
h some embodiments, the antibody that specifically binds CDS 8 is a bispecific antibody. The VL and/or the VH regions of the existing anti-CD38 antibodies or the VL and VH regions identified de novo as described herein may be engineered into bispecific full length antibodies. Such bispecific antibodies may be made by modulating the CHS interactions between the monospecific antibody heavy chains to form bispecific antibodies using technologies such as those described in U.S. Pat, No, 7,695,936; Intl. Pat. Pub) . No, WO04/111233; U.S. Pat. Publ. No. US2010/0015133; U.S. Pat. Publ. No.
US2007/0287170; Intl. Pat. Publ. No. WO2008/119353; U.S. Pat. Publ. No.
US2009/0182127; U.S. Pat, Publ. No. US2010/0286374; U.S. Pat. Publ. No.
US201 1/0123532; Intl. Pat. Publ. No. WO201 1/131746; Int. Pat. Publ. No.
WO2011/143545; or U.S. Pat. Publ. No. US2012/0149876. Additional bispecific stractures into which the VL and/or the VH regions of the antibodies of the invention may be incorporated are for example Dual Variable Domain Immunoglobulins (Inlt. Pat. Publ. No. WO2009/134776), or structures that include various dimerization domains to connect the two antibody arms with different specificity, such as leucine zipper or collagen dimerization domains (Int. Pat. Publ. No. WQ2012/02281 L U.S. Pat. No. 5,932,448; U.S. Pat, No. 6,833,441).
For example, bispecific antibodies may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parental monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Intl.Pat. Publ, No.
WO2011/131746. In the methods, the first monospecific bivalent antibody (e.g., anti- CD38 antibody) and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions may optimally be restored to non-reducing. Exemplary reducing agents that may be used are 2- mercaptoethylamine (2- MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2- carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of: 2- mercaptoethylamine,
dithiothreitol and tris(2~carboxyethyl)phosphine. For example, incubation for at least 90 min at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.
Exemplary CH3 mutations that may be used in a first heavy chain and in a second heavy chain of the bispecific antibody are K409R and/or F405L. The methods of the invention may be used to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.
Administration/ Pharmaceutical Compositions
The antibodies that specifically bind CDS 8 may be provided in the methods of the invention in suitable pharmaceutical compositions comprising the antibody that specifically bind CDS 8 and a pharmaceutically acceptable carrier. The carrier may be diluent, adjuvant, excipient, or vehicle with which the antibodies that specifically bind CD38 are administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used . These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the antibodies that specifically bind CD38 in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5%, usually to at least about 1% to as much as 15 or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21sl Edition, Troy, D.B. eel., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.
The mode of administration of the antibodies that specifically bind CD38 in the methods of the invention may be any suitable route such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art. The antibodies that specifically bind CDS 8 may be administered intratumorally, to a lymph node draining site for local delivery into the tumor using known methods.
The antibodies that specifically bind CD38 may be administered to a patient by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously or irrtraperitoneally. i.v. infusion may be given over for example 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
The dose given to a patient is sufficient to alleviate or at least partially arrest the disease being treated ("therapeutically effective amount") and may be sometimes 0.005 rng to about 100 mg/kg, e.g. about 0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 1 8, 19, 20, 21 , 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.
A fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m2. Usually between 1 and 8 doses, (e.g., 1 , 2, 3, 4, 5, 6, 7 or 8) may be administered, but 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 1 8, 19, 20 or more doses may be given.
The administration of the antibodies that specifically bind CD38 in the methods of the invention may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. For example, the antibodies that specifically bind CD38 in the methods of the invention may be administered at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every four weeks by intravenous infusion.
The antibodies that specifically bind CD38 may be administered in the methods of the invention by maintenance therapy, such as, e.g. once a week for a period of 6 months or more.
For example, the antibodies that specifically bind CD38 in the methods of the invention may be provided as a daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1 .0, 1 .1, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. The antibodies that specifically bind CDS 8 in the methods of the invention may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an e vent in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors.
The antibodies that specifically bind CD38 in the methods of the invention may be lyophiiized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.
The antibodies that specifically bind CD38 in the methods of the invention may be administered in combination with a second therapeutic agent.
Tn the methods of the invention, the antibodies that specifically bind CD38 may be administered together with any one or more of the chemotherapeutic dmgs or other anticancer therapeutics known to those of skill in the art. Chemotherapeutic agents are chemical compounds useful in the treatm ent of cancer and include growth inhibitory agents or other cytotoxic agents and include alkylating agents, anti-metabolites, anti- microtubule inhibitors, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors and the like. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and meth ylamel amines including altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethyiolomeiamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard: nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunombicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirabicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-FU; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as f!udarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid: aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil: bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidamine; mrtoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;
sizofiran; spirogermanium; tenuazonic acid; tnaziquone; 2,2',2"-trichlorotriethylamine: urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; members of taxoid or taxane family, such as pacliiaxel (TAXOL®docetaxel (TAXOTERE®) and analogues thereof; chlorambucil; gemcitabine; 6 "thioguanine; mercaptopurine; methotrexate;
platinum analogues such as cisplatin and earboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; no antrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difiuoromethylomithine (DMFO); retinoic acid;
esperami cms; capecitabine: inhibitors of receptor tyrosine kinases and/or angiogenesis, including NEXAVAR® (sorafemb), SUTENT® (sunitmib), VOTRIENT™ (pazopanib), PALLADIA™ (toceranib), ZACTIMA™ (vandeianib), RECENTTN® (cediranib), regorafemb (BAY 73-4506), axitinib (AG013736), lestaurtmib (CEP-701), TARCEVA® (erlotinib), IRESSA™ (gefitinib), i'iiloirn (afatinib), TYKERB® (lapatinib), neratinib, and the like, and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti -estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-iniidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapnstone, and FARESTON® (toremifene); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of the above. Other conventional cytotoxic chemical compounds as those disclosed in Wiemann et ai., 1985, in Medical Oncology (Calabresi et aL, eds.), Chapter 10, McMillan Publishing, are also applicable to the methods of the present invention. Exemplary agents that may be used in combination with the antibody that specifically binds CD38 in the methods of the invention include tyrosine kinase inhibitors and targeted anti -cancer therapies such as IRESSAiM (gefitinib) and Tarceva'5, (erlotmib) and other antagonists of HER2, HER3, HER4 or VEGF, Exemplar}' HER2 antagonists include CP-724-714, HERCEPTIN™ (trastuzumah), OMNTTARG™ (pertuzumab), TAK- 165, TYKERB® (lapatimb) (EGFR and HER2 inhibitor), and GW-282974. Exemplary HER3 antagonists include anti-Her3 antibodies (see e.g., U.S. Pat. Publ. No.
2004/0 97332). Exemplary HER4 antagonists include anti-HER4 siRNAs (see e.g., Maatta et al, Mol Biol Cell 17: 67-79, 2006,, An exemplar ' VEGF antagonist is
( Avastin™ (Be vacizumab) .
Exemplary agents that may be used in combination with the antibody that specifically binds CD38 in the methods of the invention include standard of care drags for solid tumors, or an immune checkpoint inhibitor.
The second therapeutic agent in the methods of the invention may be an immune checkpoint inhibitor.
In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4 antibody.
In some embodiments, the immune checkpoint inhibitor is an antagonistic anti- PD-1 antibody, an antagonistic anti-PD-Ll antibody, an antagonistic anti~PD-L2 antibody, an antagonistic anti-LAG3 antibody, or an antagonistic anti-TIM3 antibody.
In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.
In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody.
In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody.
In some embodiments, the immune checkpoint inhibitor is an anti-LAG3 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-TIM3 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.
Any antagonistic anti-PD-1 antibodies may be used in the methods of the invention. Exemplary anti-PD-1 antibodies that may be used are OPVIDO® (nivolumab) and KEYTRUDA® (pembrolizumab). OPVIDO® (nivolumab) is described in for example US. Pat. No. 8,008,449 (antibody 5C4) and comprises the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25. KEYTRUDA® (pembrolizumab) is described in for example U.S. Pat.No 8,354,509 and comprises the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23. The amino acid sequences of nivolumab and pembrolizumab are also available through the CAS registry. Additional PD-1 antibodies that may be used are described in U.S. Pat. No. 7,332,582, U.S. Pat. Publ. No. 2014/0044738, Int. Pat. Publ, No. WO2014/17966 and U.S. Pat. Publ. No. 2014/0356363.
"Antagonist" refers to a molecule that, when bound to a cellular protein, suppresses at least one reaction or activity that is induced by a natural ligand of the protein. A molecule is an antagonist when the at least one reaction or activity is suppressed by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than the at least one reaction or activity suppressed in the absence of the antagonist {e.g., negative control), or when the suppression is statistically significant when compared to the suppression in the absence of the antagonist. Antagonist may be an antibody, a soluble ligand, a small molecule, a DNA or RNA such as siRNA. A typical reaction or activity that is induced for example by PD-1 binding to its receptor PD- Li or PD-L2 may be reduced antigen-specific CD4† or CD8 cell proliferation or reduced interferon-γ (IFN-γ) production by T cells, resulting in suppression of immune responses against for example tumor. A typical reaction or activity that is induced by TIM-3 binding to its receptor, such as galectin-9, may be reduced antigen specific CD4+ or CDS" cell proliferation, reduced IFN-γ production by T cells, or reduced CD137 surface expression on CD4† or CD8" cells, resulting in suppression of immune responses against for example tumor. Hence, an antagonistic PD-1 antibody specifically binding PD-1, an antagonistic PD-L2, an antagonistic antibody specifically binding TIM-3 induces immune responses by inhibiting the inhibitory pathways.
SEQ ID NO: 22
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDM GFDYWGQGTTV TVS S
SEQ ID NO: 23
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLFIWYQQKPGQAPRLLIYLAS
YLESGVPARFSGSGSGTDFTLTISSLEPEDFA ^CQHSRDLPLTFGGGTKVEIK QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWYRQAPGKGLEWVAVIWY
DGSKRY Y AD S VKGRFT1 SRDN SKNTLFLQMN SLRAEDTA V Y Y C ATNDD Y WGQG
TLVTVSS
SEQ ID NO: 25
E1VLTQSPATLSLSPGERATLSCRASQSVSSYLA\VYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGT VEIK
Anti-PD-Ll antibodies that enhance immune response may be used in the method of the invention (e.g. antagonistic anti-PD-LI antibodies). Exemplary anti-PD-Ll antibodies that may be used are durvalumab, atezolizumab and aveiumab, and those described in, for example, U.S. Pat. Pub!. No. 2009/0055944, U.S. Pat. No. U.S. Pat, No, 8,552, 154, U.S. Pat. No. 8,217, 149 and U.S. Pat. No. 8,779,108.
Durvalumab comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 Atezolizumab comprises the VH of SEQ ID NO: 28 and the VL of SEQ ID NO:
29,
Aveiumab comprises the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31. SEQ ID NO: 26
EVQLVESGGG LVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVAN I QDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREG
GWFGELAFDYWGQGTLVTVS S
SEQ ID NO: 27
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQ PGQAPRLLIY
DASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVTYCQQYGSLPWTFG QGTKVEIK
SEQ ID NO: 28
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWG QGTLVTVS S DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLT1SSLQPEDFATYYCQQYLYHPATFGQ
GTKVEIK
SEQ ID NO: 30
EVQLLE SGGGLVQPGGSLRLS CAASGFTF S S YIMM WVRQ APGKGLEWV S S
lYPSGGnTYADTVKGRFTISRlINSKNTLYLQMNSLRAFilTAVYYCARIK
LGWTTVDYWGQGTLVTVSS
SEQ ID NO: 31
QSALTQPASVSGSPGQSrnSCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADWCSSYTSSSTRV FGTGTKVTVL
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31 for a time sufficient to treat the solid tumor.
The invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25 for a time sufficient to enhance the immune response.
The invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD 38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23 for a time sufficient to enhance the immune response.
The invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 for a time sufficient to enhance the immune response.
The invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29 for a time sufficient to enhance the immune response. The invention also provides for a method of enhancing an immune response in a patient, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD 38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-Ll antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31 for a time sufficient to enhance the immune response.
The invention also provides for a method of treating a patient having a colorectal cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-1 antibody for a time sufficient to treat the colorectal cancer.
The invention also provides for a method of treating a patient having a colorectal cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-Ll antibody for a time sufficient to treat the colorectal cancer.
The invention also provides for a method of treating a patient having a colorectal cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an an tagonistic anti-PD-L2 antibody for a time sufficient to treat the colorectal cancer.
The invention also provides for a method of treating a patient having a lung cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-1 antibody for a time sufficient to treat the lung cancer.
The invention also provides for a method of treating a patient having a lung cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-L 1 antibody for a time sufficient to treat the lung cancer.
The invention also provides for a method of treating a patient having a lung cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-L2 antibody for a time sufficient to treat the lung cancer.
The invention also provides for a method of treating a patient having a prostate cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 in combination with an antagonistic anti-PD-1 antibody for a time sufficient to treat the prostate cancer. The invention also provides for a method of treating a patient having a prostate cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD 38 in combination with an antagonistic anti-PD-Ll antibody for a time sufficient to treat the prostate cancer.
The invention also provides for a method of treating a patient having a prostate cancer, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CDS 8 in combination with an antagonistic anti-PD-L2 antibody for a time sufficient to treat the prostate cancer.
Anti-LAG-3 antibodies that enhance immune response may be used in the methods if the invention. Exemplary anti-LAG-3 antibodies that may be used are those described in, for example, Int. Pat. Pubi. No. WO2010/019570.
Anti-CTLA-4 antibodies that enhance immune response may be used in the methods if the invention. An exemplary anti-CTLA-4 antibody that may be used is ipilimumab.
Anti-PD-1, anti-PD-Ll, anti-PD-L2, anti-LAG3, anti-TlM3 and anti-CTLA-4 antibodies that may be used in the methods of the invention may also be generated de novo using methods described herein.
In some embodiments, anti-PDl antibodies comprising the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33 may be used.
In some embodiments, anti-PDl antibodies comprising the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 35 may be used.
In some embodiments, anti-TIM-3 antibodies comprising the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37 may be used.
In some embodiments, anti-TIM-3 antibodies comprising the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39 may be used.
SEQ ID NO: 32
QVQLVQSGAEVKKPGSSVKVSCKASGGTTSSYAISW^RQAPGQGLEWMGGIIPIF DTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSL
DYWGQGTLVTVSS
EIVLTQSPATLSLSPGERATLSCRASQSVRSYLA\VYQQKPGQAPRLLIYDASNRAT
GIPARFSGSGSGTDFTLTISSLEPEDFAVYl^CQQRNYAVPLTFGQGTKVEIK EVQLVESGGGLVQPGGSLRLSCAASGFAFSRYDMSWVRQAPG GLESVAYISGG
GANTYYLDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLSYFDVWG
QGTLVTVSS
SEQ ID NO: 35
E1VMTQS1 1 SVSPGERATLSCRASQSLSDYLH\¾YQQKPGQAPRL,L1KSASQS1SG IPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNGHSFPYTFGQGTKLEIK
SEQ ID NO: 36
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSG GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQ GTLVTVSS
SEQ ID NO: 37
EIVLTQSPATLSLSPGERATLSCRASQSVNDYLAWYQQKPGQAPRL-Lr DASNRAT
GIPARFSGSGSGTDFTLTISSLEPEDFA\7Y .QQGGHAPITFGQGTKVEI
SEQ ID NO: 38
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMQWRQMPGKGLEWMGAIYP GDGDIRYTQNFKGQVTISADKSISTAYLQWSSLKASDTAIVIWCARWEKST VQ RNYFDYWGQGTTVTVSS
SEQ ID NO: 39
DTQMTQSPSSLSASVGDRVTTrCKASENVGTFVSWYQQKPGKAPKLLIYGASNRY TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPTFGQGTKLEIK
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-l antibody comprising the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-PD-1 antibody comprising the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 35 for a time sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti~TIM-3 antibody comprising the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37 for a rime sufficient to treat the solid tumor.
The invention also provides for a method of treating a patient having a solid tumor, compri sing administering to the patient in need thereof a the apeutically effective amount of an antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 in combination with an anti-TIM-3 antibody comprising the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39 for a time sufficient to treat the solid tumor.
In the methods of the invention, the combination of the antibody that specifically binds CD38 and the second therapeutic agent may be administered over any convenient timeframe. For example, the antibody that specifically binds CD38 and the second therapeutic agent may be administered to a patient on the same day, and even in the same intravenous infusion. However, the antibody that specifically binds CD38 and the second therapeutic agent may also be administered on alternating days or alternating weeks or months, and so on. In some methods, the antibody that specifically binds CD38 and the second therapeutic agent may be administered with sufficient proximity in time that they are simultaneously present (e.g., in the serum) at detectable levels in the patient being treated. In some methods, an entire course of treatment with the antibody that specifically binds CD38 consisting of a number of doses over a time period is followed or preceded by a course of treatment with the second therapeutic agent, consisting of a number of doses. A recovery period of 1 , 2 or several days or weeks may be used between administration of the antibody that specifically binds CD38 and the second therapeutic agent.
The antibody that specifically binds CD38 or a combination of the antibody that specifically binds CD38 and the second therapeutic agent may be administered together with any form of radiation therapy including external beam radiation, intensity modulated radiation therapy (IMRT), focused radiation, and any form of radiosurgery including Gamma Knife, Cyberknife, Linac, and interstitial radiation (e.g. implanted radioactive seeds, GliaSite balloon), and/or with surgery.
Focused radiation methods that may be used include stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity -modulated radiation therapy (IMRT). It is apparent that stereotactic radiosurgery involves the precise delivery of radiation to a tumorous tissue, for example, a brain tumor, while avoiding the surrounding non- tumorous, normal tissue. The dosage of radiation applied using stereotactic radiosurgery may vary, typically from 1 Gy to about 30 Gy, and may encompass intermediate ranges including, for example, from 1 to 5, 10, 15, 20, 25, up to 30 Gy in dose. Because of noninvasive fixation devices, stereotactic radiation need not be delivered in a single treatment. The treatment plan may be reliably duplicated day-to-day, thereby allowing multiple fractionated doses of radiation to be delivered. When used to treat a tumor over time, the radiosurgery is referred to as "fractionated stereotactic radiosurgery" or FSR. In contrast, stereotactic radiosurgery refers to a one-session treatment. Fractionated stereotactic radiosurgery may result in a high therapeutic ratio, i.e., a high rate of killing of tumor cells and a low effect on normal tissue. The tumor and the normal tissue respond differently to high single doses of radiation vs. multiple smaller doses of radiation. Single large doses of radiation may kill more normal tissue than several smaller doses of radiation may. Accordingly, multiple smaller doses of radiation can kill more tumor ceils while sparing normal tissue. The dosage of radiation applied using fractionated stereotactic radiation may vary from range from 1 Gy to about 50 Gy, and may encompass intermediate ranges including, for example, from 1 to 5, 10, 15, 20, 25, 30, 40, up to 50 Gy in hypofractionated doses. Intensity-modulated radiation therapy (IMRT) may also be used. IMRT is an advanced mode of high-precision three-dimensional conformal radiation therapy (3DCRT), which uses computer-controlled linear accelerators to deliver precise radiation doses to a malignant tumor or specific areas within the tumor. In 3DCRT, the profile of each radiation beam is shaped to fit the profile of the target from a beam's ey e view (BEV) using a multileaf collimator (MLC), thereby producing a number of beams. IMRT allows the radiation dose to confonn more precisely to the three-dimensional (3-D) shape of the tumor by modulating the intensity of the radiation beam in multiple small volumes. Accordingly, IMRT allows higher radiation doses to be focused to regions within the tumor while minimizing the dose to surrounding normal critical structures. IMRT improves the ability to confonn the treatment volume to concave tumor shapes, for example, when die tumor is wrapped around a vulnerable structure, such as the spinal cord or a major organ or blood vessel. Subcutaneous administration of pharmaceutical compositions comprising an antibody that specifically binds CD38 and a hyaluronidase
The antibody that specifically binds CD38 may be administered as a pharmaceutical composition comprising the antibody that specifically binds CD38 and a hyaluronidase subcutaneo usly .
The concentration of the antibody that specifically binds CD38 in the pharmaceutical composition administered subcutaneous!}' may be about 20 mg/ml.
The pharmaceutical composition administered subcutaneously may comprise between about 1,200 mg - 1,800 mg of the antibody that specifically binds CD38.
The pharmaceutical composition administered subcutaneously may comprise about 1,200 mg of the antibody that specifically binds CD38.
The pharmaceutical composition administered subcutaneously may comprise about 1,600 mg of the antibody that specifically binds CD38.
The pharmaceutical composition administered subcutaneously may comprise about 1,800 mg of the antibody that specifically binds CD38.
The pharmaceutical composition administered subcutaneously may comprise between about 30,000 U - 45,000 U of the hyaluronidase.
The pharmaceutical composition administered subcutaneously may comprise about 1,200 mg of the antibody that specifically binds CD38 and about 30,000 U of the hyaluronidase.
The pharmaceutical composition administered subcutaneously may comprise about 1,800 mg of the antibody that specifically binds CD38 and about 45,000 U of the hyaluronidase.
The pharmaceutical composition administered subcutaneously may comprise about 1,600 rng of the antibody that specifically binds CD38 and about 30,000 U of the hyaluronidase.
The pharmaceutical composition administered subcutaneously may comprise about 1 ,600 mg of the antibody that specifically binds CD38 and about 45,000 U of the hyaluronidase.
The pharmaceutical composition administered subcutaneously may comprise the hyaluronidase rHuPH20 having the amino acid sequence of SEQ ID NO: 40.
rHuPH20 is a recombinant hyaluronidase (HYLENEX® recombinant) and is described in Int. Pat. Publ. No. WO2004/078140. Hyaluronidase is an enzyme that degrades hyaluronic acid (EC 3 ,2.1.35) and lowers the viscosity of hyaluronan in the extracellular matrix, thereby increasing tissue permeability.
SEQ ID NO: 40
MGVL FKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFR. PPVIPNVPFLWAWNAPS EFCLGKPDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIP
QKISLQDHLDKAKKDflWM
lELVQQQNVQLSLTEATEKAKQEFEKAG DFLVETTKLGKLLRPNHLWGWLFPD
CY HHYKKPGYNGSCFNVEI R DDLSWL TSiESTALYPSIYLNTQQSPVAATLY
VRNRVREAIRVSKIPDAKSPLPVFAYTR1VFTDQVLKFLSQDELVYTFGETVALGA
SGIVIWGTLSIMRSMKSCLLLDNYMEmr^Il Xm.AAKMCSQVLCQEQGVCIR
KNW SSDYLFiLNPDNFAIQLEKGGKFWRGKP LEDLEQFSEKFYCSCYSTLSCK
EKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLI
ISSVASL
The administration of the pharmaceutical composition comprising the antibody that specifically binds CD38 and the hyaluronidase may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. For example, the pharmaceutical composition comprising the antibody that specifically binds CD38 and the hyaluronidase may be administered once weekly for eight weeks, followed by once in two weeks for 16 weeks, followed by once in four weeks. The pharmaceutical compositions to be administered may comprise about 1,200 mg of the antibody that specifically binds CD38 and about 30,000 U of hyaluronidase, wherein the concentration of the antibody that specifically binds CD38 in the pharmaceutical composition is about 20 mg/'ml. The pharmaceutical compositions to be administered may comprise about 1,800 mg of the antibody that specifically binds CD38 and about 45,000 U of hyaluronidase. The pharmaceutical compositions to be administered may comprise about 1,600 mg of the antibody that specifically binds CD38 and about 30,000 U of hyaluronidase. The pharmaceutical compositions to be administered may comprise about 1,600 mg of the antibody that specifically binds CD38 and about 45,000 U of
hyaluronidase. The pharmaceutical composition comprising the antibody that specifically binds CDS 8 and the hyaluronidase may be administered subcutaneously to the abdominal region.
The pharmaceutical composition comprising the antibody that specifically binds CD38 and the hyaluronidase may be administered in a total volume of about 80 ml, 90 ml, 100 ml, 1 10 ml or 120 ml .
For administration, 20 mg/ml of the antibody that specifically binds CDS 8 in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, 0.04% poiysorbate 20, pH 5.5 may be mixed with rHuPH20, 1.0 mg/mL (75-150 kU/mL) in 10 mM L-Histidine, 130 mM NaCl, 10 mM L-Met ionine, 0.02% Poiysorbate 80, pH 6.5 prior to
administration of the mixture to a subject.
While having described the invention in general term s, the embodiments of the invention will be further disclosed in the following examples that should not be construed as limiting the scope of the claims.
Further embodiments of the invention
Set out below are certain further embodiments of the invention according to the disclosures elsewhere herein. Features from embodiments of the invention set out above described as relating to the invention disclosed herein also relate to each and every one of these further numbered embodiments.
1. An antibody that specifically binds CDS 8 for use in treating a patient having a solid tumor.
2. An antibody that specifically binds CD38 for use in treating a patient having a regulatory T cell (Treg) mediated disease.
3. An antibody that specifically binds CD38 for use in treating a patient having a myeloid-derived suppressor cell (MDSC) mediated disease, comprising administering to the subject a therapeutically effective amount of an antibody that specifically binds CD38.
4. An antibody that specifically binds CD38 for use in suppressing activity of a
regulatory T cell (Treg), comprising contacting the T regulatory cell with an antibody that specifically binds CD38.
5. An antibody that specifically binds CDS 8 for use in suppressing activity of a
myeloid-derived suppressor cell (MDSC), comprising contacting the MDSC with an antibody that specifically binds CD38. An antibody that specifically binds CD38 for use in treating a patient having a solid tumor, comprising reducing the number of regulatory T cells in a patient by administering the patient an antibody that specifically binds CDS 8.
An antibody that specifically binds CD38 for use in treating a patient having a solid tumor, comprising reducing the number of myeloid-derived suppressor cells (MDSC) cells in a patient by administering the patient an antibody that specifically binds CD38.
The antibody that specifically binds CD38 for use according to any of the embodiments 1-7, wherein the antibody elicits an immune response in the patient. The antibody that specifically binds CD38 for use according to any of the embodiments 1-8, wherein the immune response is an effector T cell (Teff) response.
The antibody that specifically binds CD38 for use according to any of the embodiments 1 -9, wherein the Teff response is mediated by CD4+ T cells or CDS ' T ceils.
The antibody that specifically binds CD38 for use according to embodiments 9 or 10, wherein the Teff response is mediated by CD8÷ T cells.
The antibody that specifically binds CD38 for use according to any of the embodiments 1-11, wherein the Teff response is an increase in the number of CD8+T cells, increased CD8+ T cell proliferation, increased T cell clonal expansion, increased CD8+ memory cell formation, increased antigen -dependent antibody production, or increased cytokine, chemokine or interleukin production. The antibody that specifically binds CD 38 for use according to any of the embodiments 1-13, wherein the antibody inhibits function of immune suppressor cells.
The antibody that specifically binds CD38 for use according to any of the embodiments 1-14, wherein the immune suppressor cells are regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSC).
The antibody that specifically binds CD38 for use according to embodiment 15, wherein the Tregs are ('1)3 (1)4 CD25 ( D i 27dl!" T cells.
The antibody that specifically binds CD 38 for use according to embodiment 14 or 15, wherein the Tregs express CD38.
The antibody that specifically binds CD38 for use according to any of the embodiments 13-16, wherein the Treg function is inhibited by Treg killing. The antibody that specifically binds CDS 8 for use according to any of the embodiments 17, wherein the Treg killing is mediated by antibody-dependent cell cytotoxicity (ADCC), antibody-dependent ceil phagocytosis (ADCP), cell- dependent cytotoxicity (CDC) or apoptosis induced by the antibody specifically binding CDS 8.
The antibody that specifically binds CD38 for use according to any of the embodiments 17-18, wherein the Treg killing is mediated by ADCC.
The method of claims 17-19, wherein about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of Tregs are killed.
The antibody that specifically binds CD38 for use according to embodiment 14, wherein the MDSC are CD1 i b ! !LV-DR (*D i 4 ( 1)33 ( 1) 1 5 cells.
The antibody that specifically binds CD 38 for use according to embodiment 21, wherein the CD1 lb¾LA-DROD14"CD33+CD15+ cells express CD38.
The antibody that specifically binds CDS 8 for use according to any of the embodiments 21-22, wherein the MDSC function is inhibited by MDSC killing. The antibody that specifically binds CDS 8 for use according to embodiment 23, wherein the MDSC killing is mediated by antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or apoptosis induced by the antibody specifically binding CDS 8.
The antibody that specifically binds CD38 for use according to any of the embodiments 23-24, wherein the MDSC killing is mediated by ADCC.
The antibody that specifically binds C D38 for use according to any of the embodiments 23-25, wherein about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% or 60% of MDSC are killed.
The antibody that specifically binds CD38 for use according to any of the embodiments 14-26, wherein the Teffs or the immune suppressor cells reside in bone marrow or in peripheral blood. The antibody that specifically binds CD38 for use according to any of the embodiments 14-27, wherein the solid tumor is a melanoma, a lung cancer, a squamous non-small cell lung cancer ( SCLC), a non-squamous NSCLC, a colorectal cancer, a prostate cancer, a castration-resistant prostate cancer, a stomach cancer, an ovarian cancer, a gastric cancer, a liver cancer, a pancreatic cancer, a thyroid cancer, a squamous cell carcinoma of the head and neck, carcinomas of the esophagus or gastrointestinal tract, a breast cancer, a fallopian tube cancer, a brain cancer, an urethral cancer, a genitourinary cancer, an endometriosis, a cervical cancer or a metastatic lesion of the cancer.
The antibody that specifically binds CD38 for use according to any of the embodiments 1-28, wherein the solid tumor lacks CD38 expression.
The antibody that specifically binds CD38 for use according to any of the embodiments 1 -29, wherein the antibody competes for binding to CDS 8 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5.
The antibody that specifically binds CD38 for use according to any of the embodiments 1-30, wherein the antibody binds at least to the region
SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region EKV QTLE A WV IHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
The antibody that specifically binds CD38 for use according to any of the embodiments 1 -31, wherein the antibody comprises the heavy chain
complementarity determining regions (HCDR) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) sequences of SEQ ID NOs: 6, 7 and 8, respectively.
The antibody that specifically binds CD38 for use according to any of the embodiments 1-32, wherein the antibody comprises the light chain
complementarity determining regions (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) sequences of SEQ ID NOs: 9, 10 and 11, respectively.
The antibody that specifically binds CD38 for use according to any of the embodiments 1 -33, wherein the antibody comprises the HCDR1 , the HCDR2, the HCDR3, the LCDR1 , the LCDR2, and the LCDR3 sequences of SEQ ID NOs: 6, 7, 8, 9, 10 and 11, respectively.
The antibody that specifically binds CD38 for use according to any of the embodiments 1-34, wherein the antibody comprises the VH that is 95%, 96%, 97%, 98% , 99% or 100% identical to SEQ ID NO: 4 and the VL that is 95%, 96%, 97%, 98% , 99% or 100% identical to SEQ ID NO: 5. The antibody that specifically binds CD38 for use according to any of the embodiments 1-31, wherein the antibody comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
The antibody that specifically binds CD38 for use according to any of the embodiments 1 -29, wherin the antibody comprises the HCDRi, the HCD 2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of:
a. the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b. the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c. the VH of SEQ ID NO: 8 and the VL of SEQ ID NO: 19; or d. the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
The antibody that specifically binds CD38 for use according to embodiment 37, wherein the anti-CD38 antibody comprises:
a. the VH of SEQ ID NO : 14 and the VL of SEQ ID NO : 5 ;
b. the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c. the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or d. the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21 ,
The antibody that specifically binds CD38 for use according to any of the embodiments 1-38, wherein the antibody is administered in combination with a second therapeutic agent.
The antibody that specifically binds CD38 for use according to embodiment 39, wherein the second therapeutic agent is a chemo therapeutic agent, a targeted anticancer therapy, a standard of care drug for treatment of solid tumor, or an immune checkpoint inhibitor.
The antibody that specifically binds CD38 for use according to embodiment 40, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-Li antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4 antibody.
The antibody that specifically binds CD38 for use according to embodiment 39, wherein the second therapeutic agent is administered simultaneously, sequentially or separately.
The antibody that specifically binds CD 38 for use according to any of the embodiments 39-42, wherein the patient is treated with radiation therapy.
An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor. An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor, wherein the antibody is administered in combination with the anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23.
An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor, wherein the antibody is administered in combination with the anti-PD-1 antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25.
An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in enhancing an immune response in a patient.
An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in enhancing an immune response in a patient, wherein the antibody is administered in combination with the anti-PD-1 antibody comprising the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23. An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VI, of SEQ ID NO: 5 for use in enhancing an immune response in a patient, wherein the antibody is administered in combination with the anti-PD-1 antibody comprising the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25. An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor, wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27.
An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VI, of SEQ ID NO: 5 for use in treating a patient having a solid tumor, wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29.
An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in treating a patient having a solid tumor, wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31.
An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VI, of SEQ ID NO: 5 for use in enhancing an immune response in a patient, wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27. 54. An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in enhancing an immune response in a patient, wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29.
55. An antibody that specifically binds CD38 comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 for use in enhancing an immune response in a patient, wherein the antibody is administered in combination with the anti-PD-Ll antibody comprising the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31 ,
Example 1. General materials and methods
Sample collection and processing
Peripheral blood and bone marrow aspirates were collected in heparinized tubes at baseline immediately prior to the first infusion and at specified time points during treatment. The majority of samples were evaluated using real-time flow cytometry, as they arrived at a central laboratory, 24-48 hours after collection. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood, isolated by density-gradient
centrifugation, and stored frozen until analysis. For the T-cell activation, clonality, and CD38+ Treg suppression assays, pre- and post-treatment samples were analyzed at the same time, using frozen PBMC samples.
Flow cytometric analysis of these samples was performed at BARC global central laboratory for evaluation of NK, T, B, myeloma cells (CD138T) and CD38 expression using a pre-validated immunophenotyping assay . Briefly, blood samples and bone marrow samples were stained with the following multifluorochrome antibody panels: cell lineage panel: PerCPCy5.5 -CD19 (cloneHIB 19; Becton Dickinson [BD]), APCa-CD24 (SN3; eBiosaence), PC7a-CD3 (UCHT-1; Beckman Coulter), V500a-CD16 (3G8; BD), and PEa-CD56 (MY; BD); regulatory T cell (TJeg) panel: APCa-CD25 (2A3; BD), PEa- CD127 (HIL-7R-M21 ; BD), APC-H7a-HLA-DR (G46-6; BD), and PerCPa-CD4 (L200; BD); naive/memory T-cell panel: APC-H7a-CD4 (RPA-T4; BD), PerCP-Cy5.5 -CD8 (RPA-T4 BD), PEa-CD62L (SKI 1; BD), and APCa-CD45RA (HI100; BD). CD38 expression was evaluated using Alexa 647 labeled antibody mAb 003 described in U.S. Pat. No. 7,829,693 having the VH and the VL sequences of SEQ ID NO: 14 and SEQ ID NO: 15. The blood samples were prepared using different Lyse-wash methods. For bone marrow aspirate samples either membrane or intracellular staining was performed with various antibodies. Becton Dickinson FACSLysing solution was used for lysing red blood cells in peripheral blood samples and Fix and Perm cell permeabilization reagents from Invitrogen were used for intracellular staining of bone marrow aspirate samples. Stained samples were acquired on FACS Canto II flow cytometers and data was analyzed using FacsDiva software. Absolute counts of immune cell populations in the blood samples and as percent of lymphocytes in bone marrow samples were determined at all the time points tested.
T-cel! receptor (TCR) sequencing
T-cell diversity was analyzed by deep sequencing of TCR rearrangements to assess CD8+ T-cell clonality using genomic DNA from PBMC samples. TCR sequencing was performed using Adaptive Biotechnologies commercial Immunoseq lM assay, and analysis was performed using prequalified multiplex polymerase chain reaction (PGR) assays (TR2015CRO-V-01 ), which were composed of forward and reverse primers that directly targeted the family of variable (V) genes (forward primers) and joining (J) genes (reverse primers). Each V and J gene primer acted as priming pairs to amplify somatically recombined TCRs, and each primer contained a specific universal DNA sequence.
Following the initial PCR amplification, each amplicon was amplified a second time with forward and reverse primers containing the universal sequence and adaptor sequence needed for DNA sequencing by lllumina.
T-cell responses to viral- and alloantigens
Patient PBMCs were seeded on 96 well plates (2χ 105 cells/well) and stimulated for 5 days with a cocktail of 23 major histocompatibility complex (MHC) class I-restricted viral peptides from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus (2 g/ml; CEF peptide pool ; PANATecs*) or an equivalent number of 25- Gy irradiated allogeneic PBMCs from healthy donors. Unstimulated PBMCs and PBMCs stimulated with anti-CD3/CD28-coated beads served as negative and positive controls, respectively. On day 5, interferon γ (IFN-γ) from cell-free supernatant was measured by sandwich enzyme-linked immunosorbent assay (ELISA; Human IFN gamma ELISA Ready-SET-Go; eBioscience) and served as a surrogate marker for T-cell activation.
Regulatory T-cell (Treg) suppression of effector cell functions: Carboxyfluorescein succinimidyl (CFSE) dilution assay
PBMCs from healthy donors were labelled with PerCP-Cy5.5a-CD3 (SK7; BD), KOa-CD45, (J33; Beckman Coulter), V450a-CD4 (SK3; BD), PEa-CD25 (M-A251, BD), PE Cy7a-CD127 (HIL-7R-M21 ; BD), and APCa-CD38 (ΉΒ-7; BD) and sorted by FACS Aria (BD). Sorted effector cells were labelled with carboxyfluorescein succinimidyl ester (CFSE; eBioscience) and stimulated with anti-CD3/CD28-coated beads in the presence or absence of CD38+Tregs or CD38" Tregs (1: 1 Treg to effector cell ratio) in RPMI plus 10% fetal calf serum . After 72 hours, flow cytometry was performed and the percent dilution of CFSE was used as a surrogate for T-cell proliferation.
Myeloid derived suppressor cell (MDSC) phenotyping and DARZALEX™
(daratum um ab)-mediated ADCC
PBMC from three normal healthy donors were co-cultured with myeloma tumor cell lines (RPMI8226, U266, H929) for six days, and evaluated for the production of granulocytic MDSC (G-MDSC) (CDl lb+CD14"HLA"DR:CD15+CD33+) as described in Gorgun et al. Blood 121:2975-87, 2013. G-MDSC were not present in normal healthy PBMC, however following co-culture with all three myeloma cells lines G-MDSC were present as 5- 25% of total PBMC population (data not shown). Gating strategy for flow cytometric evaluation of G-MDSC included CD l lb+ as the first gate, followed by CD 14" and HLA DR gating, and then followed by CD15+ and CD33÷ gating. G-MDSCs were cell sorted and evaluated for CD38 expression levels and sensitivity to DARZALEX IM (daratumumab) mediated ADCC. To evaluate the effect of DARZALEX™
(daratumumab) on ADCC/CDC of MDSCs, serum containing complement or an isotype control was added to ADCC assays.
Naive and memory T-ceIS analysis
Heparinized peripheral -blood samples were obtained from patients prior to each infusion of DARZALEX1M (daratumumab). Peripheral -blood mononuclear cells
(PBMCs) were isolated by Ficoll-Hypaque density-gradient centrifugation and stored in cryopreservation medium (RPMI supplemented with 10% human serum and 10% dimethyl sulfoxide) in liquid nitrogen. For FACS analysis, PBMCs were thawed and 2.x 10" cells/panel was resuspended in phosphate-buffered saline (PBS) with 0.05% azide and 0.1% HAS.
Data analysis
All data analysis and generation of relevant graphs were performed exclusively- using R software (R: A Language and Environment for Statistical Computing, R
Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2011, ISBN 3-900051 -07-0 http_//_www_R-project_org/). All treated subjects with an evaluable response were included in the data analysis. Consistently throughout, responders are defined as subjects with a Best Response per IRC of sCR, VGPR and PR, and non-responders are defined as subjects with a Best Response per IRC of MR, SD and PD
Different statistical comparisons included (i) baseline levels between responders and non-responders, (ii) baseline versus on treatment for responders and for non- responders, (iii) percent changes between responders and non-responders, (iv) ratio changes of baseline versus on treatment. Each comparison included first a test for normality with a Shapiro-Wilk test (Royston (1995) Remark AS R94: A remark on Algorithm AS 181 : The W test for normality. Applied Statistics, 44, 547-551). Almost exclusively, the data was found to not have a normal distribution. The differential level testing included conducting both a non-parametric Wilcox rank sum test (Hollander and Wolfe (1973), Nonparametric Statistical Methods. New York: John Wiley & Sons. Pages 27-33 (one-sample), 68-75 (two-sample), and a t-test following a Box Cox transformation (Weisberg, S. (2014) Applied Linear Regression, Fourth Edition, Wiley Wiley, Chapter 7). For the Box Cox transformation, a small number (le-07) was added to values equal to zero. In all cases, the two tests agreed. The Wilcox rank sum test p-values are shown in the tables throughout the specification unless otherwise indicated. When testing for differences of the baseline versus on treatment for responders and non-responders a two- group paired test per subject was ran, in all other cases a two group unpaired test was conducted.
As samples to analyze various lymphocyte populations were not taken at identical time points for the different dosing schedules, population modeling was conducted. Model fitting was done on the rank of the visits. Population modeling on total and activated NK cells involved fitting a broken stick model (Lutz et al., "Statistical model to estimate a threshold dose and its confidence limits for the analysis of sublinear dose -response relationships, exemplified for mutagenicity data." Mutation Research/Genetic Toxicology and Environmental Mutagenesis 678.2 (2009): 1 18-122.). Linear mixed effect models with random intercept and slope were fit on the B-cell, T-cell subpopulations, and leukocytes, monocytes, neutrophils and lymphocytes patient population data (Bates et al., (2014). "Ime4: Linear mixed-effects models using Eigen and S4." ArXiv e-print;
submitted to Journal of Statistical Software, http:_//_arxiv_org/abs/_1406.5823 . This linear mixed modeling was done on the relative day since treatment start (ADY). The linear mixed model fitting were done on log transformed response variables. In case of response variable values equal to zero, 0.1 was added to all response variable values to allow for modeling on log scale.
Example 2. Study 54767414MMY2002 design (S1RIUS)
The target population for Study 54767414MMY2002 (S1RIUS) is patients with advanced multiple myeloma who received at least 3 prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD) or double refractory to a PI and an IMiD. Response evaluations for the primary endpomt fmal analysis were based on assessments from an independent review committee (IRC) and computerized algorithm, using 2011 IMWG Guidelines (Clinical Study Report: An Open-label, Multicenter, Phase 2 Trial Investigating the Efficacy and Safety of DARZALEXTM (daratumumab) in Subjects With Multiple Myeloma Who Have Received at Least 3 Prior Lines of Therapy (Including a Proteasome Inhibitor and IMiD) or are Double Refractory to a Proteasome Inhibitor and an IMiD (EDMS-ERI-92399922; de Weers et al, (2011) J Immunol 186(3): 1840-1848).
These assessments included: overall response rate (OR R). duration of response, time to response and best response, clinical benefit rate, time to progression (TTP), progression free survival (PFS), and overall survival (OS).
A total of 124 subjects were treated with DARZALEXlM (daratumumab) in this study (de Weers et al, (2011) J Immunol 186(3): 1840-1848). 18 subjects were treated with 8 mg/kg and 106 subjects were treated with 16 mg/kg. The dosing schedule was as follows:
Group A: DARZALEX1M (daratumumab) 16 mg/kg: Cycles 1 and 2: Days 1, 8, 15, and 22 (weekly), Cycle 3 to 6: Days 1 and 15 (every other week), Cycles 7+: Day 1 (every 4 weeks). Each cycle was 4 weeks.
Group B: DARZALEXlM (daratumumab) 8 mg/kg: Cycle 1+: Day 1 (every 4 weeks).
The primary objective of the study was to determine the efficacy of 2 treatment regimens of DARZALE lM (daratumumab), as measured by the OR (CR + PR), in subjects with multiple myeloma who have received at least 3 prior lines of therapy including a PI and an IMiD or whose disease is double refractory to both a PI and an IMiD (Clinical Study Report: An Open-label, Multicenter, Phase 2 Trial Investigating the Efficacy and Safety of DARZ LEXlM (daratumumab) in Subjects With Multiple Myeloma Who Have Received at Least 3 Prior Lines of Therapy (Including a Proteasome Inhibitor and IMiD) or are Double Refractor ' to a Proteasome Inhibitor and an IMiD. EDMS-ERI-92399922).
The secondary objectives of this study included evaluation of the safety and to.lerabi.lity of D ARZALEX™ (daratumurnab) demonstration of additional measures of efficacy (e.g, clinical benefit, TTP, PFS, and OS) along with assessment of
pharmacokinetics, immunogenicity, pharmacodynamics, and to explore biomarkers predictive of response to DARZALEX1M (daratumurnab). Additional study related information is available from, the cl inical study protocol. (Clinical Study Report: An Open- label, Multicenter, Phase 2 Trial Investigating the Efficacy and Safety of DARZALEX™ (daratumurnab) in Subjects With Multiple Myeloma Who Have Received at Least 3 Prior Lines of Therapy (Including a Proteasome Inhibitor and IMiD) or are Double Refractory to a Proteasome Inhibitor and an IMiD. EDMS-ERI-92399922).
In Stage 1 of Part 1 , I subject (6%) responded in the 8 mg/kg group, and 5 subjects (31%) responded in the 16 mg/kg group. Therefore, only the 16 mg kg group was expanded in Stage 2 of Part 1 and in Part 2.
In the 16 mg/kg group, 31 subjects achieved response of PR or better based on IRC assessment; the ORR was 29% (95% CI: 21%, 39%). Three subjects (3%) achieved sCR, and 13 subjects (12%) achieved VGPR or better.
Example 3. Effect of DARZALEXIM (daratumurnab) on T-cell expansion and activity in patients enrolled in the 54767414MMY2002 study (SIRIUS)
CD38 is expressed on a variety of immune and hematopoietic cells. Broad immune profiling by flow cytometry was performed to examine the effect of
D ARZALEX™ (daratumurnab) on immune cell subsets and the association of baseline levels of these cells to clinical response. Various cell populations, including T-cells (CD3+, CDC, CD8+ and regulatory T-cells (Treg)), B-cells (CD 19"), NK cells, monocytes (CD14+), leukocytes, and neutrophils were e valuated by flow cytometry in peripheral blood and bone marrow aspirates at baseline and following DARZALEXlM
(daratumurnab) treatment to monitor for changes in these cellular populations in responders and non-responders.
Lymphocytes, Leukocytes, Monocytes and Neutrophils
Leukocyte, lymphocyte, monocyte, and neutrophil counts were studied in peripheral blood in responders and non-responders. Total lymphocytes were found increased with D ARZALEX™ (daratumurnab) treatment in responders with both 8 mg/kg and 16 mg/kg dose (Figure 1 ). Linear mixed effect modeling revealed an increase of 0,8 x 106 cells/μιΐ-. on log scale per 100 days (CI =: 0.06, 0.1 1 ). Slight increases were found for monocytes and leukocytes with significant increase of 0.03 x 106 cells/^iL (CI = 0.01, 0.04) and 0.03 x 106 cells/iiL on log scale (CI = 0.01, 0,05) for each 100 days respectively. Median neutrophil counts were consistent with baseline and did not vary significantly, although there was neutropenia noted for some patients.
Baseline levels of each of these cellular populations w ere compared between response groups. No evidence was found for baseline levels to be different for any of these cell types across response groups using Wilcoxon signed-rank test (Table 1).
Table L
Figure imgf000067_0001
NK cells
Total NK cells (CD 16+CD56i ) and activated NK cells (CD 16+CD56dus) were reduced with DARZALEXTM (daratumumab) treatment over time (data not shown) . B-cells
Absolute counts of B-cells (CD45XD3 CD19") were measured in peripheral blood or bone marrow aspirates during DARZALEX1M (daratumumab) treatment o ver time in responders and non-re sponders. B-cells slightly increased in the whole blood and were maintained in the bone marrow aspirates. Linear mixed modeling of B-cells in peripheral blood revealed a minimal increase of 0.1 x 10 6 ceils/μΐ [0=0.04, 0.16 for each 100 days on log scale over the course of DARZALEX IM (daratumumab) treatment. There were no changes to the percentages of B-cells (CD45+CD3"CD197Lymphocytes) in the bone marrow aspirates during daratumumab treatment, in either responders or non- responders (p = 0.1 and 0.4, respectively). Further, no evidence was found for B-cell counts to be different at baseline between responders and non-responders (p :=: 0.5).
T-cells
Lymphocytes were noted to increase with DARZALEX1J l (daratumumab) treatment (Figure 1) even though B ceils showed only a minimal increase (see above). To investigate further, various T-ce!l populations were studied (CD3 CD4+, CD8+ T cells, regulator ' T cells) in both peripheral blood and bone marrow.
CD3", CD4+ and CD8+ T-cells were increased in peripheral blood (both absolute counts/ μΐ and percentage of lymphocytes) following DARZALEX1 M (daratumumab) treatment. Figure 2 shows the percent change of absolute counts of CD31 T-cells (CD45+CD3÷) from baseline in peripheral blood over time for every- patient. The black line in the Figure shows the median absolute counts x 10e cells^L for all patients. Only visits with more than 2 observations were included into the Figure. Figure 3 shows the % change of absolute counts of CD4+ T-cells (CD45+CD3+CD4+) from baseline in peripheral blood over time for every patient. The black line in the Figure shows the median for all patients. Only visits with more than 2 observations were included into the Figure. Figure 4 shows the % change of absolute counts of CD8+ T-cells (CD45 fCD3+CD8 ) from baseline in peripheral blood over time for every patient. The black line in the Figure shows the median for all patients. Only visits with more than 2 obsen/ations were included into the Figure. Linear mixed modeling on absolute counts in peripheral blood revealed on average total T-cell (CD45†CD3 f) increase of 0.13 x 106 cells/μΐ on log scale for each 100 days (CI = 0.1, 0.15 ) following DARZALEX™ (daratumumab) treatment. CD8+ T-cells were found to significantly increase by 0.16 x10" cells/μΐ on log scale for each 100 days (CI =0.13, 0.19). CD4T cells were found to have a moderate increase of 0.11 xlO6 ceils/μΐ on log scale for each 100 days (CI = 0.09, 0.13). For each of the T-cell subpopulations, responders showed a higher maximum percent change of absolute counts to baseline than non-responders (CD3+ p:=3.2993e-05; CD4+ p=3.486e-05; CD8+ p=2.7172e-05; regulatory T cell p 0.002 ). Table 2 shows the Wilcoxon signed-rank test results for the comparison of each T-cell subpopulation in peripheral blood between responders and non-responders for percent change of absolute counts to baseline.
Table 2.
Percent change of absolute cell counts: peripheral blood
Figure imgf000069_0001
N: number of samples per group
R: responder
NR: non-responder
*responder vs. non-responder
SD: standard deviation
Similarly in bone marrow, total T-cells (CD45+CD3 ÷ as a percentage of lymphocytes) and CD8+ T-cells (CD45XD3XD8+ as a percentage of lymphocytes) were found to significantly increase during DARZALEX1M (daratumumab) treatment, for both responders and non-responders (CD3+ responders p=3.8147e-06, non-responders p=9.8225e-05; CD8+ responders p=3.8147e-06, non-responders p=0.0003). There was no change in median CD4T T-cells in either clinical response group in bone marrow. Table 3 shows the iicoxon signed-rank test results for the various T ceils as % lymphocytes in bone marrow?. Figure 5 shows the percentage (%) of CD45 rCD3+ cells over time during DARZALEXlM (daratumumab) treatment (both responders and non-responders included in the graph). Figure 6 shows the % CD45+CD3"!"CD8+ cells over time during
DARZALEXJ M (daratumumab) treatment (both responders and non-responders included in the graph).
Table 3.
Figure imgf000070_0001
Range (15.9; (18.5; 81) (24.5; (33.1; 83.3)
67.2) 79.6)
P-value* 0.00026883 3.8147e-06
N: number of samples per group
R: responder
NR: non-responder
*baseline vs. on treatment for responder or non-responder group
SD; standard deviation
While both responders and non-responders demonstrated T-cell increases in the peripheral blood and bone marrow, responders had the largest percentage change from baseline. To distinguish whether responders or non-responders had different levels of CD3+, CD4† and CD8+ T-cells prior to DARZALEX1J l (daratumumab) treatment, baseline measurements of each subgroup were compared in the peripheral blood.
There were no statistically significant differences between responders and non- responders in absolute T-cell counts at baseline in peripheral blood (Table 4) or in percentage of T cells from total lymphocytes in bone marrow (Table 5), Wilcoxon signed- rank test.
Table 4.
Absolute cell counts in peripheral blood prior to treatment at baseline
Figure imgf000071_0001
N: number of samples per group
R: responder
NR: non-responder
* responder vs non-responder per cell type
Figure imgf000072_0001
T ceils {% lymphocytes) in bone marrow prior to treatment
Figure imgf000072_0002
N : number of samples per group
R: responder
NR: non-re sponder
*responder vs non-responder per cell type
SD: standard deviation
T regulatory cells
Treg cells were identified as the CD3+CD4+CD25 "CD127dim cell population in a sample. The ratio of CDS" T cells to Tregs was assessed in the peripheral blood and bone marrow in patients treated with DARZALEX1M (daratumumab) over time. The ratio increased in both the periphery and bone marrow. Figure 7 A shows the median values of the CD8+/Treg and CD8+ CD4T cell ratios of all patients per time point in peripheral blood. Figure 7B shows the median values of the CD8+/Treg and CD87CD4" T-cell ratios of all patients per time point in bone marrow. The changes in the ratios of absolute counts of CD8+Tregs and CD8 CD4+ were significant in peripheral blood over time of treatment (Table 6) and in bone marrow (Table 7), Wilcoxon signed-rank test.
Tn a combined data analyses of SIRRJS and GEN501 study (Example 6), median ratios of CD87CD41 and CDSTTreg cells in peripheral blood were increased at week 8 (p = 5, lxl0"5 for CD87'CD4+ and p 1.8xl0"7 for CD87Treg) and at week 16 (p 0.0001 7 for CD87CD4+ and p = 4.1x10"' for CD87T'reg ). Similarly, in bone marrow?, median ratios of CD87CD4" and CD87Treg cells were increased on treatment (week 12 + 1 cycle) compared to baselme (p = 0.00016 for CD8+/CD4+ and p = 2.8x10""' for CDSTTreg). No significant differences were observed between responders and nonresponders.
Table 6.
Figure imgf000073_0001
Table 7.
T cell ratios in bone marrow
Mean
Sample N Median Range P-value*
(SD)
CD8+/CD4+(/Lymphocytes): 184.4 (32.58;
31 163.18
Baseline ( 129.5) 674.58)
Figure imgf000074_0001
Example 4. Study design (GEN501)
Study GEN501 (NCT00572488) evaluated DARZALEX™ (daratumumab) as monotherapy in double-refractory MM patients. Sample isolation, processing and statistical analyses was as described in Example 1 and Example 2, The study has been described in Lokhorst et al, N Eng J Med 373: 1207-19, 2005.
Briefly, Study GEN501 was the first-in-human clinical study of DARZALEXTM (daratumumab) in subjects with MM. It is a Phase 1/2, dose-escalation, safety study divided into 2 parts. Part 1 is an open-label, dose-escalation study; Part 2 is an open-label, single-arm study with multiple cohorts, based on the dose levels established in Part 1
In Part 1, 10 dose levels of DARZALEX 1 (daratumumab) were evaluated: 0.005, 0.05, 0.10, 0.50, 1, 2, 4, 8, 16, and 24 mg/kg. The 2 lowest dose cohorts were allocated 1 (+3) subject(s) each, and a standard 3 (+3) subject allocation was applied to the remaining 8 dose cohorts. Part 2 was an open-label, single study including two dose levels, 8 mg/kg and 16 mg/kg. Part 1 included 32 subjects and Part 2 included 72 subjects.
Example 5. DARZALEX1 M (daratumumab) treatment induces T cell clonality in patients
Given the expansion of CD8+ T-cells noted in both the periphery and the bone marrow in the MY2002 study , high throughp ut next-generation sequencing of the T-ceil receptor (TCR) was performed using the Immunoseq lM assay to determine whether expanding CD8+ T-cells were clonal in nature, indicative of an adaptive immune response. Total of 17 patient samples of subjects who were enrolled in the GEN501 study were evaluated (n=6 responders i.e., > PR; n=l 1 non-responders i.e., MR, SD, PD).
TCR sequencing revealed that DARZALEX1 M (daratumumab) treatment significantly increased clonality across patients. Figure 8 A shows the correlation between T cell clonalitv' pre- vs. post- DARZALEXlM (daratumumab) treatment (p = 0,0056). Figure 8B shows the fold change in clonalitv in individual patients. Responders are marked with the star. This data suggests that the T cell expansion noted with
DARZALEX™ (daratumumab) treatment may be clonal in nature.
Responders had a greater total expansion in the TCR repertoire (as measured by change in abundance; CIA) when compared to non -responders. Figure 8C shows the % CIA for individual patients. Group A: responders, Group B: non-responders. Statistically significant difference was observed between responders and non-responders (p=0.037). Figure 81) shows the sum of absolute change in abundance (CIA) in responders and non- responders for each expanded T cell clone. Figure 8E shows the maximum % CIA for each individual patient. Group A: responders, Group B: non-responders. Statistically significant difference was observed between responders and non-responders (p=0.048). Figure 8F shows the maximum CIA of a single T-cell clone in responders (Group A) and non-responders (Group B).
CIA was obtained by identifying significant differences in clonal abundance between two samples using Fisher's exact test (DeWitt et al. J. Virol. 2015) and summing the absolute change in abundance for each expanded clone.
Example 6. Immunomodulatory effects of DARZALEX1M (daratumumab) in patients enrolled in the GEN501 study
Various T and B cell populations were evaluated in responders and non- responders enrolled in the GEN501.
Lymphocytes
Similar to SIRIUS (MMY2002) study, lymphocytes were increased in both peripheral blood and bone marrow during DARZALEX lM (daratumumab) treatment. This increment was attributed to increased numbers of both CD4+ and CDS4 cells.
CD8+ central memory cells
CD8† T-cell phenotype was studied in patients treated with DARZALEX lM (daratumumab) over time in a subset of 17 patients enrolled in the GEN 501 study. CDS f cells from patients were identified as naive (CD45R07CD62L+) (TN) or central memory (TCM) (CD45R07CD62L+Wsh) cells using standard protocols.
Figure 9A shows the % of CD8+ nai e cells (% of CD8T cells) and Figure 9B shows the % of CDS + central memory cells. DARZALEX 1M (daratumumab) treatment significantly decreased the quantity of naive CD8" T cells (p = 1.82x lO"4 atWeek 8) and increased the quantity of CD8+ memory T cells (p = 4.88 * 10"2 at Week 8). This would suggest a transition from naive cytotoxic T cells to memory T cells which may be activated against a specific antigen. White squares indicate patients that achieved at least minimal response (> MR.) and black squares indicate patients that had stable disease or progressive disease. A significantly greater decrease in CD8+ naive T cells was apparent in patients who responded to treatment (data not shown). Figure 9C shows that
DARZALEX™ (daratumumab) treatment increased the percentage in HLA Class I- restricted T cells, which partially drive the virus-specific and alloreactive T cell responses Figure 9D shows that the expanding effector memory T cells expressed low levels of CDS 8. It is important to note that these T cells display normal and even increased functional activity against viral peptides and alloantigens (see Example 8). From these functional results we concluded that there is an expansion of, or improved activity of, antigen-experienced T cells against viral and alloantigens during DARZALEX IM (daratumumab) treatment. These data suggest that, unlike regulatory cell subsets, effector T cells do not need CD38 expression to properly function and expand.
CD38-positive Regulatory T-cells
The observation of the robust expansion and increased activity of cytotoxic T-cell together with recent literature indicating that several immune-suppressive cell subsets express CD38 prompted examination of the effects of DARZALEX™ (daratumumab) on regulatory cell populations regulatory T-cells (Tregs), myeloid derived suppressor cells (MDSCs) and regulatory B-cells (Bregs).
Regulatory T-cells (Tregs) (CD3+CD4+CD25 D 1270im) were isolated using standard protocols. The frequency of the Tregs was analyzed using flow cytometery.
A subpopulation of peripheral Tregs (10% ± 10%) expressed high levels of CD38 prior to Treg activation. Figure 10A, top panel shows the frequency of the Tregs in the CD3+CD4+ cell population (P4 cell population) at baseline. Figure 10A, bottom panel shows the subset of Tregs expressing high CD38 (PS cell population). These CD38" Treg were highly sensitive to DARZALEXllvl (daratumumab) treatment and exhibited a significant and almost immediate decline following the first dose of DARZALEX lM (daratumumab) (n = 17 patients; P = 8.88 * 10"ln at Week 1 versus baseline). The frequency of Tregs after DARZALEX™ (daratumumab) treatment is shown in Figure 10B, top panel (P4 cell population). Figure 10B, bottom panel shows that the CD38hig!l Tregs (P5 cells) was the most significantly depleted Treg population after 1st DARZALEX™ (daratumurnab) infusion. These CD38" Tregs remained depleted throughout DARZALEX™ (daratumurnab) treatment (p = 8.88>< 10~16, l . l l x lO"15, and 1.50 < 10"u at Weeks 1, 4, and 8, respectively, versus baseline. Figure IOC shows the % of CD38hl8h Tregs from total CD3 " cells at baseline, week 1 , week 4, week 8, relapse, and 6 months after the end of treatment (EOT). The CD38hlgh Tregs were recovered to the baseline at that time point. Changes in CD38+ Tregs were similar between patients who did and did not respond to treatment however, the CD8+ T-ceil:Treg ratio was significantly higher at Week 8 in patients who showed a response to DARZALEX™ (daratumurnab) (P = 0.00955; Figure 10D).
To assess the possible biological relevance of depletion of CD38 + Tregs with DARZALEX lM (daratumurnab) treatment, the suppressive capacity of CD38 f Tregs versus CD38~ Tregs on autologous CD31 T cells was assessed. In a series of experiments performed with sample from multiple heathy donors, CD38+ Tregs suppressed T-cell proliferation more robustly (9.9% cell proliferation observed) than CD38" Tregs (53.2% cell proliferation observed) or the negative control (74.9% cell proliferation observed) (Figure 10E).
Since MDSCs were not readily detectable in frozen PBMC samples, CD38" granulocytic MDSCs (CDl lb+CD14"HLA-DR"CD15+CD33+) were generated in vitro from PBMCs isolated from patients at baseline and from patients who had received one infusion of DARZALEX lM (daratumurnab). Figure 11 shows the flow cytometry histogram of identified MDSCs (Figure 11, top histogram, boxed cell population). Approximately half of the MDSCs expressed CD38 (Figure 11, middle graph; circled P7 cell population). The CD38hlgb MDSCs were nearly depleted in patients treated with DARZALEX1 M (daratumurnab) (Figure 11, bottom graph; circled P7 cell population).
The CD38high lineage nonspecific MDSCs were depleted with DARZALEX™ (daratumurnab) treatment over time in both non-responders and patients who have at least Minimal Repose to treatment. Figure 12 shows that the percentage of the CD38iuga MDSCs was reduced to nearly 0% in patients at 1 w eek.. 4 weeks or 8 weeks of treatment. The CD38mgh lineage nonspecific MDSCs returned to baseline after the end of treatment.
Patients with the largest CD38+ populations within lineage nonspecific MDSC demonstrated the best and most durable responces to DARZALEX™ (daratumurnab) treatment. Figure 13 shows that the patients 2, 4, 15, 16 and 17 having the highest percentage of CD38hlgh MDSC (as shown in Figure 1 1) and classified as patients with P or MR, had a Progression-Free Survival (PFS) of at least 8 months. The CD38high lineage nonspecific MDSCs were also sensitive to DARZALEX™ (daratumumab)-induced ADCC in vitro. ADCC assays were performed using CD38Msa MDSCs from two donors and Daudi cells as control target cells with effectontarget cell ratio of 50: 1. Figure 14 shows the results of the experiment from one donor.
DARZALEX lM (daratumumab) induced lysis of MDSC cells.
CD38+ Bregs were measured in DARZALEXTM (daratumumab)-treated patients (n = 16) and, similar to CD38 Tregs, were depleted following the first dose of
DARZALEXTM (daratumumab) (p = 0.0018 at Week 1 compared with baseline; paired Wilcoxon rank test) and remained low while patients were on treatment (Figure ISA), The FACS sorted Bregs, when stimulated, produced IL-10 (Figure 15B).
Collectively, these observations suggest that the depletion of immunosuppressive CD38+ MDSCs, Bregs, and Tregs is a significant contributor}' mechanism to
DARZALEX lM (daratumumab)-induced changes in T-cell populations and clonaiity.
Example 7. CD38+ MDSC cells are present in cancer patients
Percentage of MDSC (LinOD 14"HLADRlow/") and their CD38 expression was studied in in the peripheral blood of patients with NSCLC or prostate cancer using flow cytometry.
The percentage of MDSCs was between about 10%-37% and between about 10%- 27% of PBMCs in the analyzed samples from, the NSCLC and prostate cancer patients, respectively. CD38 expression was identified in 80-100% of Lin"CD14 HLADR"flow MDSCs from PBMCs from NSCLC patients and in 70-100% of MDSCs from PBMCs from prostate cancer patients.
Example 8. DARZALEX™ (daratumumab) enhances antiviral T-cell responses
To further assess the effect of DARZALEX llvl (daratumumab) on T-cell activation and functionality, IFN-γ production from peripheral T cells in response to viral and alloantigens was measured in DARZALEX™ (daratumumab)-treated patients (n = 7) with a range of clinical outcomes. Patients with a PR or better demonstrated significant increases in IFN-γ secretion in response to viral and alloantigens following
DARZALEX J M (daratumumab) treatment, compared with baseline, for at least one time point during treatment, suggesting that T cell function is not impaired by low CD38 expression (see Example 6, Figure9C). Similar to the TCR clonaiity data, this increase was more marked in patients who responded to DARZALEXllvl (daratumumab) than those who did not. Figure 16A shows the anti-viral response of one representative patient with VGPR. Figure 16B shows the anti-viral response of one representative patient with CR, Figure 16C shows the anti-viral response of one representative patient with PD. Figure 16D shows the anti-viral response of one representative patient with MR. In the Figures, error bars represent standard error of the mean of duplicate cultures. Asterisk denotes statistically significant changes between the indicated comparisons. Best response per Independent Review Committee is shown. Consistent with these results, vims-reactive T- cells in patients with VGPR (Figure 16E) or CR (Figure 16F) demonstrated an increase in proliferative capacity during DARZALEX™ (daratumumab) treatment.
Example 9, Mechanism of sensitivity of CD38 expressing immune cell subtypes to DARZALEX1M (daratumumab)
Data from both GEN501 and SIKH S studies indicated that some immune cells that express CDS 8 are depleted (NK ceils, regulatory T-cells (Tregs), regulatory B-ceils (Bregs), and myeloid derived suppressor cells (MDSCs)), while others that express CDS 8 increase in number (cytotoxic and helper T cells) with DARZALEX lM (daratumumab) therapy.
To address the mechanism of sensitivity, expression levels of CD38 were assessed in various subpopulations of immune cells in healthy donors and in multiple myeloma patients enrolled in either GENS 01 or SIRIUS study. Figure 17A shows a histogram of expression of CDS 8 in immune cells from a healthy donor, and Figure 17B shows a histogram of expression of CD38 in immune cells from a multiple myeloma patient. In a healthy donor, CDS 8 expression was highest on NK cells, followed by monocytes, B and T cells. In a multiple myeloma patient, CDS 8 expression was highest on plasma cells, followed by a subset of B cells, NK cells, monocytes, B -cells and T-cells. Figure 17C shows a comparison of the mean fluorescent intensity (MFI) of CD38 across NK cells, Tregs, Bregs, B- and T-cells cells from relapsed and refractory myeloma patients, demonstrating that after plasma ceils, NK cells expressed the highest levels of CDS 8, followed by regulatory T-cells (Tregs) and regulatory B-cells (Bregs).
In addition to CD38 expression, other ceil surface proteins such as complement inhibitory proteins (CIPs; CD46, CD55, CD59) may contribute to sensitivity or resistance to DARZALEX 1M (daratumumab). In vitro evaluation of CIPs across immune cell subpopulations found that NK cells express very low levels of CD59 and CD55, while other T and B cell populations express much higher levels. This could also contribute to the variability of DARZALEX lM (daratumumab) sensitivity across immune cell subtypes (data not shown). Discussion
This study describes previously unknown immunomodulatory effects of
DARZALEX™ (daratumumab) through reduction of CD38+ immune suppressive cellular populations and concomitant induction of helper and cytotoxic T-cell expansion, production of IFN-γ in response to viral peptides, and increased TCR clonality, indicating an improved adaptive immune response.
This study demonstrates that MDSCs and Bregs express CD38 and were susceptible to DARZALEX™ (daratumumab) treatment. These cells are known to be present in the tumor niicroenvironment and contribute to tumor growth, immune evasion, angiogenesis, metastasis, and production of suppressive cytokines. In addition to these CD38+ suppressive cellular subsets, a novel subpopulation of regulator}' T cells
(CD4+CD25÷CD127dim) was identified that also expressed high levels of CD38 and demonstrated superior autologous T-cell suppressive capacities. These cells were also sensitive to DARZALEX [M (daratumumab) and were significantly reduced in patients receiving treatment. DARZALEX vt (daratumumab)-mediated elimination of these CD381 immune-regulatory cells may reduce local immune suppression within the myeloma niicroenvironment and allow positive immune effector cells to expand and contribute to antitumor response.
Indeed, significant increases in broad T-ce!ls populations, including both CD4 r and CD8÷, were observed in both peripheral blood and within bone marrow (i.e., the tumor). Specific CD8" subpopulations were altered with DARZALE 1*1 (daratumumab) therapy, including significant decreases in naive T-ceils and concomitant significant increases in effector memory CD8+ T-cells, indicating a shift in effector T-ceils towards an antigenic experienced phenotype that retained immunological memory and may be reactive against tumor antigens. Ratios of CD8T:CD4+ and CD8+:Tregs also increased significantly with treatment, demonstrating a shift in positive versus negative immune regulators.
To evaluate whether expanded CD4" and CD8" T-cells were clonal in nature, the T-cell repertoire was examined in a subset of patients. T-cell clonality significantly increased with DARZALEX 1M (daratumumab) treatment, e ven in patients who had a best response of SD or who progressed. Therefore, increased T-cell clonality cannot be due simply to reduction in tumor burden. However, the skew in T-cell clonality was greater in patients with a good clinical response, and was correlated with the increase in CD8+ T- cells, suggesting the observed T-cell expansion with DARZALEX lM (daratumumab) treatment was antigen-driven. This is remarkable in this patient population, which was heavily pretreated (median of 5 prior lines of therapy ) and not expected to be able to mount a strong antitumor immune response. In addition to increased TCR clonality, patients with a response to DARZALEX™ (daratumumab) demonstrated increased T-celi responses to preexisting viral- and alloantigens, suggesting the rescue of the immune system from an immunosuppressive state.
Treatment with DARZALEX IM (daratumumab) caused a reduction in immune suppressive MDSC and regulator}- T- and B-cells. These reductions were concomitant with an expansion of CD4+ T-helper cells and CD8+ cytotoxic T-cells. T-cell clonality and functional anti-viral responses as measured by IFN-γ production also increased with DARZALEXlM (daratumumab) treatment. These observations indicate that T-cells continued to function properly, despite low CD38 expression, and suggest that increased T-cell response may be due to depletion of regulator)' cells. Further, these changes in T- cell expansion, activity, and clonality were more pronounced in patients who responded to DARZALEX iM (daratumumab) compared with those who did not. Relapse from
DARZALEX™ (daratumumab) therapy was associated with reversal of many of these changes. This suggests an additional, previously-uncharacterized mechanism of action of DARZALEX lM (daratumumab) through immunomodulation that may contribute to clinical responses and its efficacy.
Recently, antibodies that promote antitumor immune responses, rather than targeting the cancer directly, have demonstrated efficacy in a range of settings. Antibodies inhibiting CTLA-4 and PD-1 promote T-cell expansion and enhance T-celi activation, resulting in prolonged survival and delayed disease recurrence in patients with advanced solid tumors and hematologic malignancies such as Hodgkin lymphoma. By enhancing anticancer immunity, these immunomodulatorv' antibodies may not only induce clinical responses, but also prevent disease recurrence.
Example 10. Serum proieoniic analysis of multiple myeloma subjects traded with single-agent DARZALEX™ (daratumumab) in 54767414MMY2002 (SIRIUS) Part 2 clinical study
Biomarker sample collection and processing
Peripheral blood samples were collected in standard serum separator tubes (2.5 mL to 5 mL) and serum aliquots were shipped frozen SomaLogic, Inc (Boulder, CO) for miiitianalyte serum protein profiling. The serum protein profiling was performed at SomaLogic using a pre -validated SOMAscan assay that measures 1129 protein anaiytes by use of SOMAmer affinity based molecules. SOMAmer reagents are single stranded DNA -based protein affinity reagents. The assay uses small amounts of input sample (150 uL plasma) and converts the protein signal to a SOMAmer signal that is quantified by custom DNA microarray.
Each SOMAmer contains 4 functional moieties:
1. A unique protein recognition sequence
2, Biotin for capture
3 , Photocleavable linker
4. Fluorescent molecule for detection
The unique protein recognition sequence uses DNA and incorporates chemically modified nucleotides that mimic amino acid side chains, expanding the diversity of standard aptamers and enhancing the specificity and affinity of protein-nucleic acid interactions (Gold et a!., PLoS One 5:el5004, 2010) .The aptamers are selected for by SELEX. SOMAmer reagents are selected using proteins in their native conformations. As such SOMAmer reagents require an intact, tertiary protein structures for binding.
Unfolded or denatured presumably inactive proteins are not detected by SOMAmer reagents.
Master mixes of SOMAmer reagents are grouped for sample type and dilution. The reagents are pre-bound to streptavidin beads prior to sample incubation. Proteins in the samples are bound to the cognate SOMAmers during equilibrium, washed, incubated with NHS-biotin, washed and then the beads are exposed to UV light to cleave the photocleavable linker. The elution contains the SOMAmer reagents bound to their biotin labeled proteins. A streptavidin capture and subsequent washes removes the unbound SOMAmer reagents. In the final elution the SOMAmer molecules are released from their cognate proteins through denaturing conditions. The final eluate is hybridized to custom Agilent DNA microarrays and the fluorophore from the SOMAmer molecules it quantified by relative fluorescent units (RFU). The RFU is proportional to the amount of protein in the sample.
Samples from the MMY2002 study were tested in two primary batches. A first batch of 180 samples contained paired Cycle 1 Day 1 (ClDl, baseline) and C3D1 (Cycle 3 Dayl) serum samples from 90 subjects. The 180 samples were analyzed together on 3 separate SomaScan plates. The second batch of samples includes 50 ClDl samples, including 35 repeated samples from batch 1. Data analyses
Input datasets and definitions
Treated subjects with an evaluabie response were included in the data analysis. Consistently throughout the report, responders are defined as subjects with an overall best response (per IRC, for MMY2002) of sCR, VGPR, and PR, stable disease (SD) subjects as a subject with minimal response (MR) or SD, and non-re sponders are defined as subjects with an o verall best response (per IRC, for MMY2002) of progressi ve disease (PD).
Somalogic data pre-processing
Batch alignment
Batches 1 and 2 of MMY2002 samples were tested on two different versions of the SOMAscan platform. Differences between the two versions were minor, and included three SOMAmer sequences that changed between the versions (CTSE: 3594-6_l -> 3594- 6 5. FCN1 : 3613-62 1 - 3613-62 5, BMPER: 3654-27 i ·· 3654-27 4). These were removed from the analysis.
The measurements of the three batch 1 plates were aligned according to
SomaLogic's standard inter-plate calibration workflow, by defining plate-wide calibration scaling factors for each SOM Amer by calculating the ratio of a Master-mix specific global reference value to the median of 7 in-plate control calibrator measurements. The plate - specific scaling factor for each SOMAmer reagent was applied to each sample on the plate equivalently.
Given the different SOMAscan platform versions of batch 1 and 2, systematic inter-batch variability correction was done with a modified implementation of
SomaLogic's standard inter-plate calibration workflow, by leveraging the repeated measurement of 35 samples across batches. For each SOMAmer the ratio of the batch 1 post-calibration measurement divided by the batch 2 pre -calibration measurement was calculated for each of the 35 repeated samples (ri,j). The median of these 35 ratios was used to define the revised SOMAmer-specific calibration scaling factor for the batch 2 samples (ft). These calibration scaling factors were then implemented identically to the standard SOMAscan procedure. r all repealed samples = ft
Figure imgf000083_0001
Once the revised calibration scaling factors were calculated, the distributions of all the scaling factors for each batch of the analysis were plotted to assess the presence of outliers. 9 SOMAmers with extremely large or small calibrators (> 0.25 and < 3) were removed from the analyses due to poor reproducibility.
After batch alignment and SOMAmer filtering was complete for MMY2002, a log2 transformation was applied to all protein concentration values of MMY2002 to bring the data more in line with a normal distribution and to improve the performance of parametric statistical tests.
Confounding Variable Correction
Estimation of the portion of dataset variance explained by meta-variables (like demographics, response class, and sample time point) and identification of potential confounding factors was performed by principal component analysis on the centered and scaled dataset. Simple linear models were fit to identify the highest ranked PC that was significantly associated with each of the variables of interest. The significance of these associations was determined using a VVald test and the fraction of the PC variability explained by the model was estimated by the R2 of the fit. For the MMY2002 data, site ID was found to be correlated with PCI and explained the largest portion of dataset variability (> 7.37%, p-value :=: 3.71 χ 10-9). In order reduce the impact of sample acquisition site related effects within the data, ComBat28 was utilized to correct for site ID effects.
Repeated Sample Merging
The data of the 35 samples repeated between MMY2002 batches 1 and 2 was merged by calculating the mean for each protein.
Differential Protein Concentration Analysis
Responders versos non-responders
Statistical comparison of protein concentration distributions in DARZALEXTM (daratumumab) responders versus non-responders was performed at both baseline and on treatment using two complementary methods (i) Wilcoxon rank-sum test (Hollander and Wolfe, Ninparametirc Statistical Methods. New York: John Wiley & Sons. 1973. 27-33 (one-sample), 68-75 (two-sample) on each individual SOMAmer and (ii) Limma analysis (Ritchie, M/E., et al, Nucleic Acids Res 2015; 20:43(7):e47) on all SOMAmers simultaneously. All p-values were adjusted using the Benjamini-Hochberg (BH) method for multiple hypothesis correction (Benjamini and Hochberg, (1995) J. R. Statist. Soc. B.57: 289-300; R: A Language and Environment for Statistical Computing, R
Development Core Team, R Foundation for Statistical Computing, Vienna, Austria. 2011 ; ISBN 3-900051-07-0). The null hypothesis of no differential expression was rejected when the adjusted p-value < 0.05.
On treatment versus baseline
Baseline versus on treatment protein levels were compared using three alternative statistical methods: (i) two-way repeated-measures ANOVA6 (ii) the Wilcoxon signed- rank test and (iii) the Friedman test (Johnson et al, (2007) Biostatistics 8(1): 1 18-127). All p-values were adjusted to control FDR using the BH method for multiple hypothesis correction (Benjamini and Hochberg, J.R. Statist. Soc. B.57:289-300, 1995). In addition to the treatment significance, the two-way repeated-measures ANOVA (Chambers et al.. Analysis of variance; designed experiments: Chapter 5. Statistical Models in S, Editors J.M Chambers and T.i Hastie. Wadsworth & Brookes/Cole. 1992) was also applied to determine if significant time-point: response-class interaction occurred for each
SOMAmer. A modified Wilcoxon rank-sum test was applied as a post-hoc test to specifically determine if responders and non-responders showed different treatment effects, by calculating the difference between every subject's on treatment and baseline protein concentration values and performing a Wilcoxon rank-sum test. Significance values were adjusted using the BH method and the null hypothesis was rejected when the adjusted p-value < 0.05.
Classifier training
Baseline protein level MMY2002 data was used to build a response prediction classifier. A nested-loop stratified 10-fold cross-validation approach repeated 30 times, using 4 different machine learners: Support Vector Machines (SVM), Random Forests (RF), Naive Bayes (NB), and j48 decision trees. For each learner, the training procedure began with creating 10 balanced folds of the dataset (outer loop). One of these folds was held out as a test cohort while the remaining 9 were passed to an inner loop as the training cohort. Within the inner loop, the training cohort was once again split into 10 balanced folds, creating inner-training and inner-test sets. Learners were trained on each of these inner-training sets and this process was repeated 30 times for each cohort within the outer loop. The accuracy of each inner loop learner at predicting the inner-test sets was used to select features and optimize model parameters. Once the 3 Ox inner looping was complete for each training cohort, the performance of the outer loop (using the optimized paramete and features) was assessed on each corresponding test cohort. The entire outer looping procedure was then repeated 30 times, producing 30 response predictions for every sampl within the dataset. The AUC, Sensitivity, and Specificity statistics obtained from this looping approach were an approximation of how well the final model, trained on the full original dataset, will perform on new test cases.
Results from MMY2002 study
Various comparisons were conducted including treatment induced response dependent changes in protein expression. One of the proteins that showed decreased expression in responders ove time was PD-L1, whereas PD-L1 protein expression increased in non-responders over time. The engagement of PD-L1 on T cells leads to reduced T cell function and increased Treg development. Figure 18 shows protein expression profile of PD-L1 in responders, non-responders and in patients with stable disease at cycle 1 and cycle 3.
PD-L1 engagement with its receptor PD-1 suppresses anti-tumor responses and drives T cell anergy and exhaustion. While not wishing to be bound by any particular theory, downregulation of PD-L1 upon CD38 treatment may also result in improved potentiation of anti-tumor immune responses in solid tumors.

Claims

We Claim:
1) A method of treating a patient having a solid tumor, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds CD38 for a time sufficient to treat the solid tumor.
2) The method of claim 1, wherein the antibody that specifically binds CD38 elicits an immune response in the patient.
3) The method of claim 2, wherein the immune response is an effector T cell (Teff) response.
4) The method of claim 3, wherein the Teff response is mediated by CD4+ T cells or CD84' T cells.
5) The method of claim 4, wherein the Teff response is mediated by the CD8+ T cells.
6) The method of claim 3, wherein the Teff response is an increase in the number of the CD8T T cells, increased CD8+ T cell proliferation, increased T cell clonal expansion, increased CD8+ memory ceil formation, increased antigen-dependent antibody production, increased cytokine production, increased chemokine production or increased interleukin production, (by which cells)
7) The method of claim 1, wherein the antibody that specifically binds CD38 inhibits function of an immune suppressor cell.
8) The method of claim 7, wherein the immune suppressor cell is a regulatory T cell (Treg).
9) The method of claim 8, wherein the Treg is a CD3+CD4+CD25+CD127dim T ceil.
10) The method of claim 9, wherein the Treg expresses CD38.
11) The method of claim. 10, wherein the Treg function is inhibited by killing the Treg.
12) The method of claim 1 1 , wherein killing the Treg is mediated by antibody-dependent cell cytotoxicity (ADCC).
13) The method of claim 7, wherein the immune suppressor cell is a myeloid-derived suppressor cell (MDSC).
14) The method of claim 13, wherein the MDSC is a GD I ! b ILADR"CD14"CD33+CD15 cell.
15) The method of claim 14, wherein the CDl lb+HLADR"CD14"CD334'CD15 cell expresses CD38.
16) The method of claim 15, wherein the MDSC function is inhibited by killing the MDSC,
17) The method of claim 16, wherein killing of MDSC is mediated by ADCC. 18) The method of claim 7, wherein the immune suppressor cell is a regulatory B cell (Breg).
19) The method of claim 18, wherein the Breg is a CD19iCD24+CD38 + cell.
20) The method of claim 19, wherein the Breg function is inhibited by killing the Breg,
21) The method of claim 20, wherein killing the Breg is mediated by ADCC .
22) The method of claim 7, wherein the immune suppressor cell resides in bone marrow or in peripheral blood.
23) The method of any of the claims 1-22, wherein the solid tumor i s a melanoma, a Sung cancer, a squamous non-small cell lung cancer (NSCLC), a non-squamous NSCLC, a colorectal cancer, a prostate cancer, a castration-resistant prostate cancer, a stomach cancer, an ovarian cancer, a gastric cancer, a liver cancer, a pancreatic cancer, a thyroid cancer, a squamous cell carcinoma of the head and neck, a carcinoma of the esophagus or gastrointestinal tract, a breast cancer, a fallopian tube cancer, a brain cancer, an urethral cancer, a genitourinary cancer, an endometriosis, a cervical cancer or a metastatic lesion of the cancer.
24) The method of any of the claims 1-23, wherein the solid tumor Sacks detectable CDS 8 expression,
25) The method of any of the claims 1-24, wherein the antibody that specifically binds CD38 is a non-agonistic antibody.
26) The method of claim 25, wherein the non-agonistic antibody induces proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner.
27) The method of any of the claims 1-26, wherein the antibody that specifically binds CD38 competes for binding to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5.
28) The method of claim 27, wherein the antibody that specifically binds CD38 binds at least to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region
EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
29) The method of any of the claims 1-28, wherein the antibody that specifically binds CD38 comprises a heavy chain complementarity determining region (HCDR) 1, a HCDR2, a HCDR3, a light chain complementarity determining region (LCDR) 1, a LCDR2 and a LCDR3 amino acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10 and 11 , respectively. 30) The method of any of the claims 1-29, wherein the antibody that specifically binds CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
31) The method of any of the claims 1-26, wherein the antibody that specifically binds CD38 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1 , the LCDR2 and the LCDR3 of:
a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c) the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
32) The method of claim 31 , wherein the antibody that specifically binds CD38 comprises: a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c) the VH of SEQ ID NO: 18 and the VI, of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
33) The method of any of the claims 1-32, wherein the antibody that specifically binds CD38 is administered in combination with a second therapeutic agent.
34) The method of claim 33, wherein the second therapeutic agent is a chemotherapeutic agent, a targeted anti -cancer therapy, a standard of care drug for treatment of solid tumor, or an immune checkpoint inhibitor.
35) The method of claim 34, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4 antibody.
36) The method of claim 35, wherem the immune checkpoint inhibitor is an anti-PD-1 antibody.
37) The method of claim 36, wherein the anti-PD-1 antibody comprises
a) the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23;
b) the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25;
c) the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33; or
d) the VH of SEQ ID NO: 34 and the VL of SEQ ID NO:35.
38) The method of claim 35, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.
39) The method of claim 38, wherein the anti-PD-Ll antibody comprises
a) the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27;
b) the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29; or
c) the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31. 40) The method of claim 35, wherein the immune checkpoint inhibitor is an anti-PD-L2 antibody.
41) The method of claim 35, wherein the immune checkpoint inhibitor is an anti-LAG3 antibody,
42) The method of claim 35, wherein the immune checkpoint inhibitor is an aiiti-TIM-3 antibody.
43) The method of claim 42, wherein the anti-TlM-3 antibody comprises
a) the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37; or
b) the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39.
44) The method of any of the claims 33-43, wherein the second therapeutic agent is
administered simultaneously, sequentially or separately.
45) The method of any of the claims 1-44, wherein the antibody that specifically binds CD38 is administered intravenously.
46) The method of any of the claims 1-44, wherein the antibody that specifically binds CD38 is administered subcutaneously in a pharmaceutical composition comprising the antibody that specifically binds CD38 and a hyaluronidase.
47) The method of claim 46, wherein the hyaluronidase is rHuPH20 of SEQ ID NO: 40.
48) The method of any of the claims 1-47, wherein the patient is treated or has been
treated with radiation therapy.
49) The method of any of the claims 1-47, wherein the patient has had or will undergo surgery.
50) A method of suppressing activity of an immune suppressor cell, comprising contacting the immune suppressing cell with an antibody that specifically binds CD38.
51) The method of claim 50, wherein the immune suppressor cell is a Treg.
52) The method of claim 51 , wherein the Treg is a CD3+CD4+CD25+CD127dim T cell.
53) The method of claim 50, wherein the immune suppressor cell is a MDSC.
54) The method of claim 53, wherein the MDSC is a CD1 lb†HLADR"CD14"CD33+CD15" cell.
55) The method of claim 50, wherein the immune suppressor cell is a Breg.
56) The method of claim 55, wherein the Breg is a CD19+CD24+CD38† cell.
57) The method of any of the claims 50-56, wherein the antibody that specifically binds CD38 is a non-agonistic antibody.
58) The method of claim 57, wherein the non-agonistic antibody induces proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner. 59) The method of any of the claims 50-58, wherein the antibody that specifically binds CD38 competes for binding to CD38 with an antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
60) The method of claim 59, wherein the antibody that specifically binds CD38 binds at least to the region SKRNIQFSCKNIYR (SEQ ID NO: 2) and the region
EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
61) The method of any of the claims 50-60, wherein the antibody that specifically binds CD38 comprises the HCDR1 , the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 ammo acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10 and 11, respectively.
62) The method of claim 61 , wherein the antibody that specifically binds CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
63) The method of any of the claims 50-58, wherein the antibody that specifically binds CD38 comprises the HCDR1, the HCDR2, the HCDR3, the LCDRl , the LCDR2 and the LCDR3 of:
a) the VH of SEQ ID NO : 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c) the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
64) The method of claim 63, wherein the antibody that specifically binds CD38 comprises: a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c) the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
65) A method of enhancing an immune response in a patient, comprising administering to the patient an antibody that specifically binds CD38.
66) The method of claim 65, wherein the patient has a cancer or a viral infection.
67) The method of claim 65 or 66, wherein the antibody that specifically binds CD38 is a non-agonistic antibody.
68) The method of claim 67, wherein the non-agonistic antibody induces proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner.
69) The method of any of the claims 65-68, wherein the antibody that specifically binds CDS 8 competes for binding to CDS 8 with an antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5. 70) The method of any of the claims 65- 69, wherein the antibody that specifically binds CD38 binds at least to the region S RNIQFSCIYR (SEQ ID NO: 2) and the region EKVQTLEAWVMGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).
71) The method of any of the claims 65-70, wherein the antibody that specifically binds CD38 comprises the HCDR1, the HCDR2, the HCDR3, the LCDRl , the LCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10 and 1 1, respectively.
72) The method of any of the claims 65-70, wherein the antibody that specifically binds CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
73) The method of any of the claims 65-68, wherein the antibody that specifically binds CD38 comprises the HCDR1, the HCDR2, the HCDR3, the LCDRl, the LCDR2 and the LCDR3 of:
a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c) the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
74) The method of claim 73, wherein the antibody that specifically binds CD38 comprises: a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 1 7.
c) the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
75) A method of treating a patient having a viral infection, comprising administering to the patient an antibody that specifically binds CD38 for a time sufficient to treat the viral infection.
76) The method of claim 75, wherein the antibody that specifically binds CD38 is a non- agonistic antibody.
77) The method of claim 75 or 76, wherein the non-agonistic antibody induces
proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner.
78) The method of any of the claims 75-77, wherein the antibody that specifically binds CD38 competes for binding to CD38 with an antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
79) The method of any of the claims 75-78, wherein the antibody that specifically binds CD38 binds at least to the region SKRNIQFSCIYR (SEQ ID NO: 2) and the region EKVQTLEAWVMGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1 ). 80) The method of any of the claims 75-79, wherein the antibody that specifically binds CD38 comprises the HCDRl, the HCDR2, the HCDR3, the LCDRl, the LCDR2 and the LCDR3 amino acid sequences of SEQ ID NOs: 6, 7, 8, 9, 10 and 11, respectively.
81) The method of any of the claims 75-80, wherein the antibody that specifically binds CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5.
82) The method of any of the claims 75-77, wherein the antibody that specifically binds CD38 comprises the HCDRl, the HCDR2, the HCDR3, the LCDRl, the LCDR2 and the LCDR3 of:
a.) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 1 7.
c) the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
83) The method of claim 82, wherein the antibody that specifically binds CD38 comprises: a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15;
b) the VH of SEQ ID NO: 16 and the VL of SEQ ID NO: 17;
c) the VH of SEQ ID NO: 18 and the VL of SEQ ID NO: 19; or
d) the VH of SEQ ID NO: 20 and the VL of SEQ ID NO: 21.
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